Metal coordinated compositions

ABSTRACT

A metal coordination complex of a biologically active moiety and a metal is disclosed. The complex confers to the biologically active moiety an improved performance which can include potency, stability, absorbability, targeted delivery, and combinations thereof.

This application is a continuation of non-provisional patent applicationSer. No. 11/257,504 filed on Oct. 24, 2005, which claims priority fromprovisional application 60/621,747, filed Oct. 25, 2004.

FIELD OF THE INVENTION

This invention relates to novel metal coordinated complexes ofbiologically active molecules.

BACKGROUND OF THE INVENTION

It is desirable to improve the properties of known, biologically activemolecules by modifying their structures. The goal of such modificationsis a molecule that is improved in some way, such as potency, stability,reduced side effects, or targeted delivery. This improvement is achievedwithout sacrificing the molecule's desirable properties. While this goalis easily stated, it is difficult to achieve in actual practice, as theeffects of any particular modification is often highly unpredictable.

SUMMARY OF THE INVENTION

According to the current invention, the structure of known biologicallyactive molecules is modified to result in new molecules known as metalcoordinated complexes. These new molecules have unexpectedly superiorproperties. The metal coordinated complexes of the current inventioninclude complexes of thyronine, tetracycline antibiotics, oxycodone andhydrocodone, and complexes of their derivatives.

Chelation is a critical component in the stabilization of a metalcoordinated complex. For the s-block metals, this is particularly truefor calcium and magnesium. For example, it can be seen that the logK_(eq) of the acetic acid-magnesium complex is 0.47. With theincorporation of a single amino group on the molecule (i.e., glycine)the log K_(eq) increased to 1.34. Magnesium typically prefers chelationwith oxygen over nitrogen and this effect can be seen by comparing thelog K_(eq) of adenine (log K_(eq)=2.08) with that of 6-hydroxypurine(log K_(eq)=6.65). Magnesium forms particularly strong bonds withoxidized phosphorus, such as phosphates, as is revealed by comparing thelog K_(eq) of adenosine (log K_(eq)=0.50) with that ofadenosine-5′-monophosphoric acid (log K_(eq)=1.80).

In general, zinc complexes are more stable then the comparable magnesiumcomplexes. This is particularly true if the ligand bears nitrogen orsulfur. (This may not be the case for ligands with oxygen only and evenless so if the ligand is a phosphate.) Using the glycine example above,the log K_(eq) for the glycine-zinc complex is 4.85. The strength of thezinc sulfur bond versus the oxygen bond is manifest in the relative logK_(eq) values for the zinc complexes of hydroxypropanoic acid (logK_(eq)=0.86) and mercaptopropanoic acid (log K_(eq)=6.43). Comparisonsof log K_(eq) values with other metals and ligands reveal that thischelation stabilization prevails in metal coordination chemistry.

Whereas it may not be required that chelation occur to form a stablemetal coordinated complex with inherent covalency, and this isparticularly true with the transition metals combined with nitrogenousligands, in most cases it is a preferred embodiment of this inventionthat the active agent chelate with the metal, particularly if the metalis magnesium.

It is an embodiment of this invention that the active agents that makethe best candidates for complexing with magnesium and calcium are thosethat have a proton on a heteroatom (i.e., oxygen, nitrogen or sulfur)with a pK_(a) slightly greater than water or lower than water and havean additional heteroatom in close proximity to the first protonatedheteroatom such that it can participate in the bonding, or otherwisechelate, with the metal. Drugs that have this arrangement of functionalgroups are most likely going to bond with a metal, where the resultantmetal coordinated active agent will be stable enough in a biologicalsystem and survive hydrolysis therein, such that the performance of theactive agent will be sufficiently modulated. This hydrolytic stabilityimparted by multidentate ligands is supported by the fact that they canlower the pK_(a)'s of the ligand such that even amides can bedeprotonated with weak bases, such as triethylamine, in the presence ofcoordinating metals. Therefore, active agents with protons onheteroatoms, which normally would not be ionized in typical biologicalpH, can have the proton replaced with a covalently coordinated metal,where covalency is enhanced by the additional chelation fromparticipating heteroatoms. It is a preferred embodiment of thisinvention that at least one of the heteroatoms on the active agent thatwill bind to magnesium or calcium be oxygen or sulfur. Magnesium formsunusually strong bonds with phosphates and phosphonates and, therefore,it is an additional embodiment of this invention that the active agentcoordinated with magnesium is an organophosphate or organophosphonatecompound.

It is an embodiment of this invention that the active agents that makethe best candidates for complexing with zinc and the p-block metals arethe same as those with the s-block metals with the additionalflexibility that if the active agent has two nitrogens, a nitrogen and amercaptan or two mercaptans in a proper chelation arrangement, then thepresence of a proton on a heteroatom is not necessary to form a stablemetal coordinated complex. It is a further embodiment of this inventionthat transition metals have further ligation flexibility in thatchelation is even less required for their covalent coordinationcomplexes if the ligands have at least one nitrogen or mercapto group.

The active agents which are embodied in this invention can be dividedinto chemical classes as shown in Table 1 (actually they may be dividedinto combinations of chemical classes to reflect the heterogenouschelation potential). The drugs listed in Table 1 are not intended to bean exhaustive list of all drugs that satisfy the embodiment of thisinvention but a representation of the chemical classes that exist inpharmaceuticals and that other pharmaceuticals that are of the sameclass listed in Table 1 or have arrangements of atoms that is satisfiedby the embodiments of this invention are also claimed by this invention.

TABLE 1 Biologically active molecules that form coordination complexs inaccordance with the Invention. Chemical Class or Functional GroupCombination Therapeutic Classes Drug Examples Guanide or diamineAntidiabetic, AntiGERD, Metformin, Famotidine, Antineoplastic,Antiviral, Mitoxantrone, Adefovir, Antihypertensive Hydralazine,Zanamivir Amine or amide with GERD, Diuretic, Famotidine, sulfonamideAntimigraine, Antidiabetic Hydrochlorothiazide, Sumatriptan, Glipizde,Glyburide, Torsemide Amine or amide with azole GERD, Antiviral,Lansoprazole, antimigraine, Antiurolithic, Zolmitriptan, Rabeprazole,Antihypertensive, Omeprazole, Analgesic, Anitemetic Esomeprazole,Ribavarin, Allopurinol, Clonidine, Granisetron Amine or amide withalcohol Antineoplastic, Antiviral, Mitoxantrone, Saquinavir, Boneresorption inhibitor, Alendronate, Albuterol, Antibiotic,Bronchodilator, Ephedrine, Epinephrine, Antithrombotic, Analgesic,Dipyramidole, Oxycodone, Antihypertensive, Oxymorphone, Anxiolytic,Anticonvulsant Tetracycline, Minocycline, Doxycycline, Labetalol,Lorazepam, Oxazepam β-diketone, α-diketone, Antibiotic, Antineoplastic,Tetracycline, Minocycline, ketophenol, α-ketoalcohol Antiinflammatory,Doxycycline, β-ketoalcohol Multiple sclerosis Mitoxantrone, Atovaquone,treatment Betamethasone, Paclitaxel, Docetaxel, Methylprednisolone,Prednisone, Idarubicin β-ketoacid Antibiotic Levofloxacin, Ofloxacin,Norfloxacin Ureide Antiviral, Tenofovir, Acyclovir, Antiparkinsonian,Cabergoline, Theophylline, Bronchodilator Valgancyclovir Amine or amidewith acid Antihypertensive, Quinapril, Ramipril, Hormone replacement,Trandolopril, Enalipril Antiparkinsonian, Diuretic, Lisinopril,Thyroxine, Antipsoriatic, Liothyronine, DOPA, Antineoplastic,Furosemide, Methotrexate, Antirheumatic, Antibiotic, Penicillin,Amoxicillin, Antiepileptic, Cefotetan, Captopril, Antidepressant,Analgesic Gabapentin, Ketorolac Alcohol with azole Angiotensin IIreceptor Losartan, antagonist, Phosphonate or phosphate Bone resorptioninhibitor, Alendronate, Etidronate, Antiviral FosamprenavirPhosphonateor phosphate with Antiepileptic Fosphenytoin amide Diol orpolyol Bronchodilator, Nutritional Albuterol, Epinephrine, supplements,Contrast Myoinositol, Chiroinositol, imager Iodixanol Mercaptan withacid Antiasthmatic, Antibiotic Montelukast, Cefazolin, CefotetanMercaptan with amine or Antipsychotic, Olanzapine, Captopril amideAntihypertensive Amine with amide Hormone deficiency, Tabimorelin,Amoxicillin, Antibiotic Loracarbef, Iodochlorohydroxyquin Alcohol withacid Analgesia, Cholesterol Salicylic acid, lowering, AntihypertensiveAtorvastatin, Mesalamine, Antiinflammatory Pravastatin, Sitofloxacin,Trovafloxacin Dicarboxylic acid Antineoplastic Pemetrexed Amine withN-oxide Antialopecia agent Minoxidil Alcohol with Nitrites AntibioticMetronidazole Diene with alcohol, amine, Antiacne, AntineoplasticRetinoic acid, Fenretinde amide or acid Oligonucleotide (polyureide Genetherapy, Anti-AMD iRNA, Pegaptanib or polyphosphate) Oligopeptide(polyamide) Immunosuppressant, Cyclosporin, Epoetin, Antianemic,Antiviral, Inteferon, Atrial Natriuretic Antineoplastic, DiureticPeptide, Abarelix Oligosaccharide (polyol) Anticoagulant, Heparin,Acarbose, Antidiabetic, Antibiotic Gentamycin, Tobramycin GERD =Gastroesophageal Reflux Disease AMD = Age-related Macular Degeneration

BRIEF DESCRIPTION OF THE DRAWINGS

For the present invention to be clearly understood and readilypracticed, the present invention will be described in conjunction withthe following figures, wherein:

FIG. 1 illustrates the structure of Magnesocene in accordance with theprior art.

FIG. 2 illustrates the structure of(Cyclopentadienyl)-^(t)butylmethylbis(N,N′-[2,6-diisopropylphenyl]amidinate)magnesiumin accordance with the prior art.

FIG. 3 illustrates the structure of magnesium:salicylaldehyde complex inaccordance with the prior art.

FIG. 4 illustrates the structure of Magnesium phthalocyanine inaccordance with the prior art.

FIG. 5 illustrates an outer sphere RNA:magnesium coordination complex inaccordance with the prior art.

FIG. 6 illustrates an inner sphere RNA:magnesium coordination complex inaccordance with the present invention.

FIG. 7 illustrates an RNA:magnesium:arginine coordination complex inaccordance with the present invention.

FIG. 8 illustrates a substituted arginine:magnesium complex inaccordance with the present invention.

FIG. 9 illustrates salicylic acid and polymer bound arginine complexedwith magnesium in the inner sphere and peptides encapsulating theligand:metal complex in the outer sphere in accordance with the presentinvention.

FIG. 10 illustrates a magnesium:oxycodone complex in accordance with thepresent invention.

FIG. 11 illustrates the proton NMR ofBis(triiodothyroninato)-bis(dimethylsulfoxide)magnesium in accordancewith the present invention.

FIG. 12 illustrates the proton NMR of Triiodothyronine (T3) inaccordance with the prior art.

FIG. 13 illustrates the proton NMR of Bis(triiodothyroninato)zinc inaccordance with the present invention.

FIG. 14 illustrates the proton NMR of Dimethylbiguanide:Zinc complex inaccordance with the present invention.

FIG. 15 illustrates the proton NMR of Dimethylbiguanide in accordancewith the prior art.

FIG. 16 illustrates the proton NMR of Tetracycline in accordance withthe prior art.

FIG. 17 illustrates the proton NMR of Bis(tetracyclinato)magnesium inaccordance with the present invention.

FIG. 18 illustrates the proton NMR of Tetracycline-magnesium complexwith 1N HCl added in accordance with the present invention.

FIG. 19 illustrates the proton NMR of Tetracycline with 1N HCl added inaccordance with the present invention.

FIG. 20 illustrates the proton NMR of Hydrochlorothiazide in accordancewith the prior art.

FIG. 21 illustrates the proton NMR of Hydrochlorothiazide-Zinc complexin accordance with the present invention.

FIG. 22 illustrates the proton NMR of Hydrochlorothiazide-Zinc complexwith 1N HCl added in accordance with the present invention.

FIG. 23 illustrates the proton NMR Bis(acycloguanosinato)magnesium inaccordance with the present invention.

FIG. 24 illustrates the structure ofBis(triiodothyroninato)-bis(dimethylsulfoxide)magnesium in accordancewith the present invention.

FIG. 25 illustrates the structure of Bis(triiodothyroninato)zinc inaccordance with the present invention.

FIG. 26 illustrates the structure of Bis(minocyclinato)magnesium inaccordance with the present invention.

FIG. 27 illustrates the structure of Bis(tetracyclinato)magnesium inaccordance with the present invention.

FIG. 28 illustrates the structure of Dimethylbiguanide-zinc complex inaccordance with the present invention.

FIG. 29 illustrates the structure of Bis(acycloguanosinato)magnesium inaccordance with the present invention.

FIG. 30 illustrates the relative pharmacokinetic profile of T3, T3Mg,T3Zn in a rat animal model in accordance with the present invention.

FIG. 31 illustrates the IEF profiles of magnesium and zinc iRNAcomplexes in accordance with the present invention. The rows labeled “A”refer to complexes prepared in anhydrous conditions. The rows labeled“W” refer to complexes prepared in water.

DETAILED DESCRIPTION OF THE INVENTION

Chemical bonds exist in three basic forms: ionic, covalent andcoordination or the so-called Werner complexes, which are typicallylarger than inorganic metal salts. (It should be pointed out that Wernercomplexes are considered to have neutral ligands.) The differencesbetween the three bond types can be attributed, in part, to thethermodynamic stability of the bond, particularly in solution.Conversely, the stability of a compound can be expressed as thepropensity for the atoms of the molecule to separate or dissociate insolution.

The thermodynamic stability of a compound is expressed in terms of itfree energy of formation according to equation 1:

ΔG=−RT ln K  Equation 1

Where ΔG is the Gibbs free energy and indicates the thermodynamicstability of the compound. The more negative ΔG is the more stable thecompound. R is the gas constant, T is the absolute temperature and K isthe equilibrium constant. The equilibrium constant is expressed as aratio of products over reactants. In the case of coordination compoundsfor the reaction:

M+xL

ML _(x)

K is expressed in equation 2:

K=[ML _(x) ]/[M][L] ^(x)  Equation 2

Thus the increasing thermodynamic stability of a compound is directlyrelated to the increasing value of the equilibrium constant.

In certain cases it is advantageous to express the equilibrium constantin terms the dissociation potential of a metal-ligand bond. The reactionis thus:

ML _(x)

M+xL

The dissociation constant, K_(diss) is shown in equation 3:

K _(diss) =[M][L] ^(x) /[ML _(x)]  Equation 3

Whereas it is commonly accepted that ionic bonds nearly completelydissociate in solution, most covalent bonds, do not dissociate at all.Thus, determining bond strength in solution, through measurement of thedissociation constant or the more commonly expressed parameter theequilibrium constant, is a method of discerning the bond type. Forcoordination compounds, which involve bonding between metals and ligandsfrom Groups 15-17, the thermodynamic stabilities have not been firmlyestablished.

Examination of the literature reveals that covalency of organometallicbonds can be determined from spectroscopic data (i.e. NMR and MS), abinitio molecular mechanics calculations or a combination of the two. Ingeneral, covalency is most likely to occur with transition metals, withnitrogen and sulfur ligand atoms (in preference to oxygen) and withincreasing bond order or haptivity (designated “η”) from the ligand.Organometallic compounds with ligands that having multiple haptivitiesare described as chelates. Amongst the metals in groups 1 and 2, the socalled s-block main group elements, only beryllium and magnesium areconsidered to be important chelate forming elements.

Recent research, using ab initio theoretical calculations, has furtherqualified the nature of the coordination bond in terms of the ionic vs.covalent nature of the ligand-metal bond. Pierloot applied the CASSCF(complete active-space self-consistent field) model to a series ofWerner complexes to measure the degree of covalency of theseorganometallic complexes. Her general conclusions were that a trendexists wherein the static correlation energy, obtained from the CASSCFcalculations, correlates well with covalency of the metal-ligand bond.She further concluded, that for the same metal, the metal-ligandcovalency and related correlation effects increase in the followingorder of ligands:

F′<OH₂<NH₃<<Br⁻<I⁻.

This was in agreement with the nephelauxetic effect described byJorgensen. The magnitude of this effect was directly correlated to thereduction of the interelectronic repulsion of a transition metal uponcoordination in a ligand field. This reduction depended on the ligandsand was expressed as the ratio of the Racah parameter, B, in the complexand in the free metal ion (β=B_(complex)/B_(ion)). The reduction in Bresulted from a decrease in electron-electron repulsion of the freemetal ion after ligands were added to form the metal complex; a largereduction in B indicates a strong nephelauxetic effect. Thus ionicligands, such as F⁻, give a small reduction in B and have larger βvalues. Based on spectroscopic measurements, ligands were orderedaccording to decreasing β values generating the nephelauxetic series:

F′>OH₂>(NH₂)₂CO>NH₃>H₂NCH₂CH₂NH₂≈(COO)₂ ²⁻≈(CO₃)²⁻>NCS⁻>Cl⁻>CN′>Br⁻>N₃⁻>I⁻>S²⁻≈(C₂H₂O)PS₂ ²⁻>diarsine

The correlation of the two series is supported by the similaritiesbetween the effects that both techniques described. In the former case,the CASSCF calculation gauges the contribution of the metal d-orbital tothe metal-ligand bond. Racah parameter reduction by complex formation(B_(complex)) is caused by delocalization of the transition metald-orbital electron cloud on the ligands, which is indicative of covalentbond formation.

Bonding between Li⁺ and Be²⁺ with Cp ligands is mostly ionic due to thelow energy state of the contributing metal bond relative to the Cp bond.In addition, the ionic radius of these elements is too small to allowmore than one Cp ligand to bond.

Theoretical calculations of magnesocene (FIG. 1), Cp₂Mg, reveal that thestructure of the compound resembles that of Cp₂Ca, Cp₂Sr and Cp₂Ba butthe d-orbital populations of Mg were found to be negligible in Cp₂Mg.However, the Mulliken charge for Mg in Cp₂Mg using the densityfunctional theory (DFT) model predicted 0.66; a value close to 2 isexpected for a compound with a large dissociation constant such asMgCl₂. This is in agreement with a paper by Faegri, Almlöf and Lüthi,who, according to ab initio MO-LCAO calculations conclude that thecharge separation of magnesocene is only slightly higher than that offerrocene (Cp₂Fe), a known covalent coordination compound. These datawould suggest that the Mg—Cp bond is somewhat covalent. The Cp moietycontributes its covalent bonding partly from its negative charge andpartly from Π-bonding from the double bonds. This combination of anionicand Π-bonding with metals would also occur in retinoic acid and itsanalogs. Thus forming metal coordinated compounds of retinoic acid is anembodiment of this invention.

The reactivity of Cp₂Mg and related Mg—Cp compounds was studied byWinter, et. al., and they found that magnesium forms stable bonds withamidinate ligands. Perhaps most telling was the stability of[CpMg(η²-^(t)BuC(N(2,6-^(i)Pr₂C₆H₃))₂)], which was sublimed unchangedwith 80% recovery at 180° C./0.05 torr (FIG. 2). Thus an example of astable magnesium-amidinate compound has been reported, which providesfurther support to the covalency of such compounds. This is importantfor compounds that contain amidinate functionalities such as the purineand arginine containing compounds.

Whereas it is commonly accepted that transition metallocenes have strongcovalent character, a legitimate argument that main group metalloceneshave significant covalent bond elements has been made as well. The lackof d-orbital participation in the metal-ligand bonding may reduce thestability of the compound but does not preclude the notion that maingroup metals, particularly magnesium, can form bonds with ligands thatare more covalent than ionic. It is generally known that the formationof 6-coordinate magnesium complexes upon their crystallization is due tosp³d² hybridization. So it is conceivable that in certain situationseven the d-orbitals of magnesium can participate in bonding of thecoordination complex.

One can see a large difference in stability when comparing theequilibrium constants of a Werner complex with a metal halide. Forexample, log K_(Mg-pyridine)-=2.08 and log K_(MgCl2)≈−1.0. Keeping inmind that pyridine is a neutral ligand; this difference in log K canonly be due to the covalency of the magnesium-pyridine bond vis-à-visthe ionic nature of the magnesium-chloride bond. Another example of astable magnesium complex is seen with magnesium-salicylaldehyde (SA)complex, with a log K_(Mg-SA2)=6.80 (FIG. 3). The stability of this bondis remarkable in that the ligand bonding atom is oxygen, which typicallytend to form ionic bonds with metals. However, the existence ofchelating oxygen stabilized the complex beyond what a pure ionic bondwould do. Although nitrogen will form stronger bonds to magnesium thancalcium, typically oxygen is a stronger chelator of magnesium thannitrogen.

The equilibrium constants of chelates are typically very large (e.g.,log K_(eq) for magnesium ethylenediamine-N,N′-disuccinate complex is6.09) and may not reveal the extent of covalency between the neutralpart of the ligand and the metal. However, it is the equilibriumconstant that dictates the stability of any coordination compound andthat is an important criterion for determining the nature of thechemical entity and how it will perform in particular applications. Theexistence of a covalent bond within the complex and its contribution tothe stability of chelates can explain their very large log K_(eq) andmay also contribute to the rigidity of the molecular structure. Itshould be pointed out that, in many cases, covalency is the mostimportant contributor to the stability of a coordination complex.

The magnesium porphyrin complexes or chelates are likely the most wellknown organomagnesium compounds; chlorophyll is a magnesium porphine.Phthalocyanine is a porphyrin representing the basic elements of thatclass of compound and is used extensively as a model system to studymetal-porphyrin bonds. It has been determined that transition metalsform complexes with phthalocyanine (FIG. 4) very easily but becausealkali and alkali earth salts dissociate so completely in water andother protic solvents, no solvent has been found, so far, which issuitable for direct introduction of Li⁺, Na⁺, K⁺, Sr²⁺ and Ba²⁺ fromsolutions of their salts. As predicted from the ease of complexation ofMg²⁺ and Be²⁺, only these two s-block elements along with Ca²⁺, can bedirectly introduced into phthalocyanine, which is typically done fromtheir iodide or perchlorate salts in pyridine.

The synthesis, structure, stability and physical properties ofmetal-porphyrin complexes have been well studied. The structure and thephysical properties of magnesium phthalocyanine have been furtherelucidated using a variety of techniques, most recently, near-IRabsorption and X-ray crystallography. The recurring conclusion is thatthe magnesium-porphyrin chelate represents an extremely stable exampleof a metal coordinated compound.

Certain magnesium-ligand complexes are indeed covalent in nature and notionic and thus are new compositions of matter and not merely new saltforms. Metal-organic ligand compounds, covalency in the nature of theirbonds.

It is an embodiment of this invention that the formation of acoordination complex is favored when the ligand has direct bondingopportunity to the inner sphere of the metal, preferably magnesium. Thisis accomplished by using anhydrous magnesium and non-protic solvents (orif the solvent is protic it should be bulky). This concept is supportedby the fact that the catalytic reactivity of a metal ion is reduced inits hydrated form. Complex formation in aqueous systems is a delicatebalance between hydrogen bonds between ligand and water and thecompetition for binding sites on the metal by hydration and complexationcapability of the ligand. It follows that complexation of a ligand withthe inner sphere of metal is also reduced in aqueous systems. It furtherfollows that the converse is true—that is, the rate of chelation orcomplexation of metals with ligands in non-aqueous systems isaccelerated vis-à-vis aqueous systems.

A composition comprising an organic active agent bound to a metal as astable metal-ligand coordination compound with inherent covalency is asa new molecular entity. In another preferred embodiment of theinvention, the metal is selected from the main group elements. In yet afurther embodiment of the invention, the metal is selected from thes-block elements. In a preferred embodiment of the invention, the metalis magnesium.

Furthermore, it is an embodiment of this invention that virtually anydrug-magnesium complex with a K_(eq)>1.0 has enough inherent stabilityto modulate the pharmacokinetics of dissolution, absorption,distribution, metabolism and excretion. Given that the dissociationconstant of Mg(OH)₂ is −11.5, it is not surprising to discover that mostmagnesium complexes are much more stable in alkaline conditions than inacid. Thus the stability of the drug-magnesium complex in the smallintestines is likely to modulate the pharmacokinetics of drugabsorption. For those metal-drug complexes that are acid labile, it isan embodiment of this invention that protection of the complex from theacidic milieu of the stomach be accomplished by a coating orencapsulation material that releases the complex upon entry into thesmall intestines. It is a further embodiment of the invention that theencapsulation agent is a ligand or group of ligands forming an outercoordination sphere.

Another important concept of this invention is that simple combinationsof metal with ligands in solution do not always produce the sameproduct. It is recognized that several, if not many, patents claimvarious salts as dependent claims without any support in the subjectmatter. This is accepted because the salt of an organic acid is easilyprepared by treating it with a base and a metal salt where the expectedproduct is the metal salt of the organic acid; a method known by anyoneskilled in the art. However, when coordination chemistry contributes tothe bonding between the organic acid and the metal, a variety ofconditions, such as solvent, temperature and, perhaps most importantly,ligands attached to the metal, impact the structure and the stability ofthe coordination complex.

Additional ligands, other than the drug, can stabilize the metal-drugcomplex. For example the K_(eq) of the glycine (G) magnesium bond is1.34. If, however, salicylaldehyde is added to the complex, theequilibrium for the reaction

Mg²⁺+SA⁻+G⁻

Mg(SA)(G)

is 4.77. Clearly salicylaldehyde adds a stabilizing effect to magnesiumglycine bond. It is an embodiment of this invention, that adjuvants,like salicylaldehyde are incorporated into the drug: metal complexes toimpart beneficial physicochemical properties. It is a further embodimentof this invention that the benefit of adjuvants is to stabilize thedrug: metal complex in certain environments, such as in aqueoussolutions.

There are very few examples of coordination complexes with transitionmetals found in the Physician's Desk Reference (“PDR”) and include 1)insulin modified by zinc; 2) carboplatin contains platinum; 3) niferexis a polysaccharide-iron complex; 4) pyrithione zinc, used as the activeingredient in anti-dandruff shampoo. In addition, some nutritionalsupplements are described as complexes. Chromium picolinate is oneexample, where three picolinic acid groups are bound to a single Cr⁺³ inan octahedron (the nitrogens provide the three other binding sites).Whereas it is an embodiment of this invention that the metal is selectedfrom the group representing transition metals in a more preferredembodiment of the invention the metal is selected from the s-block maingroup elements, groups 1 and 2. In a most preferred embodiment of theinvention the metal is magnesium.

The patent literature cites some examples of novel magnesium coordinateddrugs, which include: 1) Trilisate® a stable, solid choline magnesiumsalicylate composition mentioned above for treating arthritic pain; 2)magnesium salts of2-descarboxy-2-(tetrazol-5-yl)-11-desoxy-15-substituted-omega-pentanorprostaglandinsimparting greater tissue specificity and ease of purification andcompounding into medicaments; 3) magnesium vanadate with insulinomimeticproperties with utility in treating insulin resistance syndromes; 4) acrystalline magnesium-taurine compound for treatment of thrombotic orembolic stroke and prophylactic treatment of pre-eclampsia/eclampsia andacute cardiac conditions; 5) the magnesium omeprazole “salt” derivativesmentioned above to treat GERD.

The science of pharmaceuticals salts is a well studied area andselection of the salt form can impact a given pharmaceutical'sperformance. Examples of effects that the salt form can have on a druginclude dissolution rate, solubility, organoleptic properties,stability, formulation effects, absorption modulation andpharmacokinetics. The periodical, Drug Delivery, published an article inOctober, 2003, citing three excipient applications using metals,presumably forming salts, to stabilize pharmaceutical agents. HumanGrowth Hormone is complexed with zinc to reduce its hydrophilicity andthereby slow the drug release; stabilization of proteins against theacidic environment produced by degradation of encapsulating polymers wasaccomplished by adding magnesium hydroxide to the formulation; zinccarbonate was used to stabilize vinca alkaloids from acid hydrolysis.Whereas these products clearly use metals to stabilize thepharmaceutically active agent, the latter two do not claim to havemodified the structure of the active agent.

Further it is well established that by simply changing salt forms, thepharmacokinetics of absorption in the small intestines is significantlymodulated. For example, the chemical structures of both the phosphatesalt of tetracycline and tetracycline hydrochloride differ in thatportion of the salt form which is not the pharmacophore and one wouldexpect that the relative physical properties of each would not have agreat influence on their relative bioavailabilities. But in fact, thephosphate salt is absorbed twice as much as the hydrochloride salt.Conversely, the bioavailability of the free acid of warfarin is nearlyequivalent to its sodium salt, which is unexpected because thedissolution rate of the warfarin sodium tablet is 350 times faster thanthe tablet containing the free acid. If different salt forms can confersuch changes in the kinetics of absorption, than a complex with anS-block main group element may have even a more pronounced effect onabsorption.

It is known that the bioavailability of tetracycline antibiotics ismainly influenced by the physicochemical properties of their metalcomplexes that will most likely form in the GI tract. This is clearly anindication of drug:metal bond formation in vivo. The formation of acovalent bond between a drug and a metal in vivo is even a morereasonable expectation when the drug contains nitrogen and the metal isin Groups 10-12 (e.g., nickel, copper, zinc). It is an embodiment ofthis invention that modulation of a drug's performance is imparted by aformulation of the drug and the metal, which will facilitate formationof a stable complex between the drug and the metal.

It is an embodiment of this invention that the following benefits can beconferred upon a drug when complexed or coordinated with a metal:

-   -   1. Improved water solubility, which can equate to better        bioavailability (see discussion below);    -   2. Enhanced lipophilicity for improved absorption through the        cell membrane;    -   3. Locking the pharmacophore into a conformation for improved        receptor binding;    -   4. Ameliorating formulation problems due to polymorphism (see        discussion below);    -   5. Acid absorption properties for protection from degradation in        acidic environments, such as the stomach;    -   6. The stability of the coordination complex may infer a delay        in absorption of the active pharmacophore. This is important for        drugs like liothyronine, where rapid absorption of the drug        increases toxicity potential.    -   7. Bioadhesion properties for sustained absorption of the active        pharmacophore;    -   8. Prevent abuse of narcotic analgesics by binding the        pharmacophore of the narcotic through an organometallic complex        to render the narcotic inert unless ingested.

Nature provides many examples of how transition metals are transported,stored and utilized. Perhaps the most well known example is hemoglobin,which is iron porphyrin. As stated earlier chlorophyll is a porphinestructure surrounding magnesium. Some enzymes require metals in orderfor them to be active. That is the reason why trace metals, such ascopper, zinc, chromium, etc. are important for proper nutrition. Evensome antibodies have transition metals associated with them. The metalis required for enzyme activity due to the metal locking the peptidestructure of the enzyme in a conformation through the formation of acoordination complex.

The concept of incorporating computer aided design of drugs has gainedpopularity in recent years. This technique, which has been referred toas in silico, has developed to the point that through the understandingof allosteric, coulombic and non-covalent interactions between thesubstrate and the receptor, lead drug candidates have been identified bycomputer modeling, before any material has been produced. It is anembodiment of this invention that by including metal coordination in thecomputer simulated molecule, new and improved lead compounds can beidentified. It is a further embodiment of this invention that in silicoderived lead compounds will have altered docking thermodynamics when theincorporation of a metal as a complex of the lead compound is includedin the calculations. It is a yet further embodiment of this inventionthat compounds previously removed from consideration based onunsatisfactory in silico analyses will become important lead compoundswhen reanalyzed with the incorporation of a metal complex into thecalculations. It is a preferred embodiment of this invention that themetal used for the revised in silico calculations as described above areselected from the main group elements. In a more preferred embodiment ofthis invention the metal is selected from the s-block main groupelements. It is a preferred embodiment of this invention that the metalis magnesium.

In terms of direct application of the complex to a biological system, itis an embodiment of this invention that active agents that requireligand-receptor binding are imparted enhanced biological activity byvirtue of the active agent's conformational structure being locked inplace through complexation with a metal. The receptor can bemembrane-associated, within the cytoplasm or circulating in the body. Itis an embodiment of this invention that metals be incorporated intoinjectable drugs to lock the drug into a conformation that providesoptimum interaction with its target receptor. It is a preferredembodiment of this invention that the metal be considered safe forinjection. It is yet an even further embodiment of this invention thatthe metal be selected from the list of aluminum, bismuth, magnesium,calcium, iron or zinc. It is yet a further preferred embodiment of thisinvention that the active agent is selected from the list of injectabledrugs, including, but not limited to, vaccines, antineoplastics,antidiabetic drugs, and antisense RNA or other metabolic modulators.

The metal coordination technology of this invention could also advancecurrent research in vaccine design. For example, a new cancer vaccinebeing developed combines a lipoprotein adjuvant, a peptide antigen witha carbohydrate antigen specific for cancer cells. The three componentsof the vaccine construct are joined together covalently via linkers.This method of constructing the vaccine is common in bioconjugatechemistry. Metal coordination can be used as a scaffold to bind thedifferent components of a bioconjugate such as Pegaptanib, whosecombined components are an aptamer, polyethylene glycol and a lipid. Itis an embodiment of this invention that the components of a bioconjugatecan be combined in a single molecular entity by complexing eachcomponent to a central metal. It is a further embodiment of thisinvention that metal coordination serves as a general technique inbioconjugate chemistry.

Of particular note is the remarkable affinity that magnesium has fornucleic acids. With the advent of antisense RNA, interference RNA andaptamers as therapeutic agents it will be increasingly important toincorporate delivery technologies for these nucleic acid drugs. Some ofthe drug delivery techniques that are currently being investigatedinclude pegylation, liposomes or anionic clays. Interestingly, a recentstudy released by Howard Hughes Medical Research Lab indicated thatmontmorillonite clay facilitated entry of RNA into lipid vesicles. Thereare a variety of clays that vary in the amount of alumina, silica,magnesia, iron and potassium. Thus, forming a magnesium complex of RNAmay facilitate RNA's entry into vesicles, which are considered to be alaboratory model of cellular membranes.

The efficacy of the magnesium-nucleic acid complex can be evaluatedvis-à-vis the nucleic acid alone using in silico techniques describedabove. Thus it becomes an important embodiment of this invention thatnucleic acid drugs' efficacy is enhanced by their coordination withmetals. It is a further embodiment of this invention that the nucleicacid be combined with a metal to form a coordination complex prior toadministration. A significant portion of the complexes in simplecombination of a metal salt with a nucleic acid in aqueous systems willbe outer sphere coordinated ligands (FIG. 5) and may not provide theoptimum conformation for receptor binding, particularly for membranetransport applications. A major premise of this invention is thatmetal-ligand complex structure is impacted significantly if the ligandhas the opportunity to be an inner sphere ligand (FIG. 6) in preferenceto being an outer sphere ligand.

It is a further premise of this invention that inner sphere ligandformation is promoted by using anhydrous conditions to prepare themetal-ligand complex. It is an embodiment of this invention; therefore,that the metal ligand complex is prepared under anhydrous conditions andthat reconstituting the complex in water will produce a coordinationcomplex with greater covalency, greater stability, greater cellpermeability and modulated biological performance relative to thecomplex prepared in water. This system is very amenable to incorporatingadjuvants, such as polyarginine to enhance transfection efficiency,within the inner coordination sphere (FIG. 7).

Perhaps the most important development in recent times in gene therapywas the discovery and advanced research on interference RNA (“iRNA”).Unlike antisense RNA, iRNA is recycled by the cell's biochemicalmachinery to further silence gene products encoded by mRNA. This resultsin increased efficiency of the gene silencing. The major problemsassociated with iRNA include their permeability into cells and theirstability, particularly in the presence of nucleases.

These problems have been addressed by the incorporation of pulmonarysurface active material (“SAM”), lipid or amine based transfectionagents, electroporation, viral vectors or plasmid vectors. The lattertechnique is particularly interesting in that the plasmid vectors causethe siRNA to adopt a “hairpin” structure and these iRNA variants havebeen given the name of short hairpin RNA or shRNA. These shRNA moleculeshave enhanced silencing capacity. Moreover, there is a body of evidencethat suggests that transfection agents are not necessarily required forthe siRNA molecules to enter the cell. The recent discovery of pulmonaryapplications of siRNA and viroids are two reported phenomena whereinnaked RNA can enter the cell and silence gene products. As a matter offact, it is well known by scientists in this field that the secondarystructure of the RNA does not seem to impact its gene silencing effects.

RNA is an oligonucleotide with multiple phosphate groups. Magnesiumforms very strong bonds with phosphates and so RNA-Mg complexes arelikely to have the magnesium atoms bound to the phosphate groups. Bycombining magnesium with RNA under anhydrous conditions, a covalent bondis formed, which, theoretically, would increase the lipophilicity ofthat portion of the RNA molecule. Furthermore, the magnesium center canbind multiple phosphate groups, theoretically, causing the formation ofthe hairpin structure mentioned above. This hairpin structure would notonly manifest a lipophilic residue but would also provide greaterresistance to attack from nucleases, which would lead to greaterstability.

Since RNA would have other phosphate groups in excess of what is boundto magnesium, that portion of the RNA molecule would retain its watersolubility. This novel form of RNA would have the desired amphiphilicproperties that are important for mass transfer (hydrophilicity) andabsorption (lipophilicity). For further discussion on this point see the“Improved solubility” section below.

A typical process would entail combining RNA with a magnesium salt in ananhydrous solvent. A suitable solvent may be DMSO or perhaps an ionicliquid. An advantage of ionic liquids is that recovery of themagnesium-RNA complex would merely entail adding the solution to anionic liquid miscible non-solvent such as alcohol (or in some casessupercritical CO₂ may work), where the desired product would precipitateout. The ionic liquid could then be recycled for the next reaction bydistilling off the alcohol.

The above process would likely be applicable to any water solublebiologically active agent. Thus it is an embodiment of the inventionthat the biologically active agent is any saccharide, peptide ornucleotide. In a preferred embodiment of the invention the biologicallyactive agent is a nucleotide. In a more preferred embodiment of theinvention the biologically active agent is an antisense RNA,interference RNA or an aptamer. It is a preferred embodiment of theinvention that the metal is selected from the main group elements. It isa further preferred embodiment of the invention that the metal isselected from the s-block main group elements. It is recognized thatmagnesium binds to nucleic acids more tightly than calcium, thus it is amost preferred embodiment of the invention that the metal is magnesium.

Improved Solubility/Permeability

In quantifying drug absorption it is useful to apply the termbioavailability. This is defined as the fraction (F) of the dose thatreaches the systemic circulation. Thus, in the extreme cases, F=0 indrugs which are not absorbed at all in the GI tract while for drugs thatare completely absorbed (and not metabolized by a first pass effect)F=1. The bioavailability can be calculated from the area under the curve(AUC) of the serum level vs. time plot. It depends on many factors andsome of these factors differ between normal individuals. In terms ofbioavailability, drugs have been classified into four categoriesaccording to the table below.

Class Solubility Permeability Bioavailability Expectation I High HighVery high bioavailability but is rare due to the requirement for activetransport II Low High Reasonable bioavailability if solubility problemis not too severe III High Low Low permeability is difficult to overcomeand drugs may be shelved for this reason. IV Low Low Very low or nobioavailability. Drugs in this class are usually not developed anyfurther.

As can be seen a delicate balance between cell membrane permeability andsolubility needs to be struck for a drug to become a viable candidatefor further development. The reason for this is because physicalproperties that enhance solubility (i.e. hydrophilicity) are usuallyorthogonal to those properties enhancing permeability (i.e.hydrophobicity or lipophilicity).

The interaction between metals and tetracycline antibiotics has beenshown to reduce the bioavailability of both the drug and the metal. Asstated earlier, the bioavailability of tetracycline antibiotics aremainly influenced by the physicochemical properties of the metalcomplexes that prevail in the GI tract. Electric charge has the greatestimpact on bioavailability since neutral species are more likely toreadily absorb into the phospholipid membrane of the intestinal cells. Alipophilic metal coordinated complex should serve to allow greaterbioavailability vis-à-vis metal salts of the drugs, which carry electriccharges. Thus it is an embodiment of this invention that byadministering lipophilic metal-antibiotic covalent complexes,physicochemical properties of the antibiotic can be controlled and,further, may prevent the metals in the GI tract to impact the dynamicsof metal interaction with the drug and ultimately absorption. It is afurther embodiment of this invention that the above stated principle isgenerally applicable to all drugs.

This technology can also be used to increase the lipophilicity of highlywater soluble drugs, or the so called Class III drugs. In this case, theconversion of an ionic center, such as a phosphate or sulfate group, isconverted to a covalent bond. This change in bonding between metal andligand is known to decrease water solubility and increase organicsolvent solubility or lipophilicity of the ligand.

If a drug is poorly soluble but is readily permeable one way itssolubility can be enhanced is by covalently attaching water solubleentities such as amino acids or carbohydrates, to the drug.Alternatively, by forming a metal-ligand complex between the drug and anionized metal center a new chemical entity is formed that now hasinherent hydrophilicity imparted to it. It is an embodiment of thisinvention to bind the active agent to a transition metal or alkalineearth metal to form a new composition of matter that has improvedsolubility while retaining its permeability. Since the new metal ligandbond is covalent, it is preferable that the metal have additionalligands (e.g., amino acid) attached to it to counterbalance thelipophilic nature of the newly formed covalent metal center.

Due to the covalent nature the stability of the metal-active agentcomplex is retained up to transport to the water film coating of thebrush border membrane. When the complex reaches the membrane the metaland the drug are separated by the lipids in the membrane accepting thelipophilic active agent and rejecting the hydrophilic metal. This isimparted through physicochemical action and, in contrast to the earliermethods of increasing solubilities of drugs, does not require enzymes.

Drugs are applied to the skin to elicit an effect to the 1) skinsurface, 2) an effect within the stratum corneum, 3) an effect requiringdeeper penetration into the epidermis and dermis or 4) a systemic effectthrough penetration to the vasculature. The aim of this research is todesign a new transdermal drug delivery (TDD) system that will allowpenetration of the drug through the epidermis or into the vasculature.The desired level of penetration will depend on the drug.

The stratum corneum provides an effective barrier and prevents water andchemicals from penetrating to the epidermis and beyond. It has beenproposed that the structural organization of the lipids in the stratumcorneum is an important factor in preventing fast transport of water andchemicals. This organization of lipids results in a liquid crystalmorphology and penetration though this matrix is caused bydestabilization of the liquid crystal through a disordering of the lipidhydrocarbon chains. This is the mechanism that has been proposed for thehydrotropes' ability to enhance penetration of topically applied drugs.

Some of the classes of chemicals that are used to enhance skinpermeability include alcohols, alkyl methyl sulfoxides, pyrrolidones,surfactants (anionic, cationic and nonionic), and fatty acids andalcohols. In addition, laurocapram, urea, calcium thioglyclate, acetoneand dimethyl-m-toluamide have been used to enhance skin penetration ofspecific bioactive reagents. Most of these drug vehicles' effect is byvirtue of their hydrotropic properties. In chemical terms, many of themhave a large dipole moment; that is they have a lipophilic portion and ahydrophilic portion. It is this large dipole moment which is a majorcontributing factor that causes these chemicals to disorder the lipidsin the stratum corneum.

Many drugs do not intrinsically possess enough skin penetrative abilityto be used topically. Thus, virtually every topically appliedpharmaceutical requires a formulation that includes a vehicle or TDDenhancer in order to achieve the desired efficacy. Aside from thestandard requirements of safety and efficacy to which allpharmaceuticals must comply, topically applied drugs need to be solubleand stable in the vehicle, the formulation must have content uniformity,the formulation must have proper viscosity and dispersioncharacteristics and must maximize patient compliance, which means itmust not be uncomfortable to apply, have an unpleasant odor or causeskin irritation.

Most notably, the lag time for the drug's penetration into theepidermis, which relies on its ability to partition from the vehicleinto the stratum corneum, has presented significant obstacles during thedevelopment of TDD formulations. Previous reports show that this lagtime can be anytime between minutes to several days. Thus, a majorimpediment of the development of TDD systems has been these additionalconsiderations unique to this application and, historically, thedevelopment time for transdermal pharmaceuticals has often been viewedas exorbitant.

Enhanced transdermal permeability of a drug complex according to thisinvention relies on the stability of the complex coupled with itsamphiphilic properties. Thus it is an embodiment that the formation of acovalent metal-drug bond converts the drug into an effective hydrotropecapable of enhancing TDD of the drug itself. It is a further embodimentof the invention that, if a TDD enhancer is still required, the metalwill act as an anchor for the vehicle and the entire complex will behaveas a single molecular entity. The advantage with this is that drugrelease from the complex no longer requires differential partitioncoefficients between the vehicle and the lipid matrix of the epidermis.

Due to its covalent nature, the stability of the metal-active agentcomplex is retained should be retained during transport through thestratum corneum. When the complex is in the epidermis the metal and thedrug are separated by the lipids in the membrane accepting thelipophilic active agent and rejecting the hydrophilic metal. This isimparted through physicochemical action and, in contrast to the earliermethods of increasing solubilities of drugs, does not require enzymes.

Converting a drug to a metal coordination complex also facilitates entryinto the eye. It has been shown that converting sulfonamides fortreating intraocular pressure (IOP) to their metal coordinationcomplexes increased their IOP reduction effect. It is believed that thisis due, in part, to the increased presence of the sulfonamide in the eyeand that this, in turn, is due to the right balance between lipo- andhydrosolubility of the metal coordinated complex. Drugs to treat eyediseases can be improved by converting them into a metal coordinationcomplex according to this invention. This is very important to treatage-related macular degeneration (AMD), where the current therapies relyon injection of the drug behind the eye. An eye drop application of adrug to treat AMD greatly improves patient compliance; coordinating theAMD drug with a metal accomplish this.

Controlling Polymorphism

Polymorphism contributes a significant portion to the variability indosages in part due to variation in solubility. Historically speaking,an inherent physical property of organometallic compounds is that stablecrystalline forms are relatively easy to prepare. Thus it is a furtherembodiment of this invention that polymorphism is overcome by convertingthe active agent into a metal complex and subjecting the complex torecrystallization processes by methods commonly known by those skilledin the art. In so doing the active moiety is “locked” into a desiredpolymorph.

Modulating Drug Absorption

In recent times there has been a flurry of activity to improve drugs bymodulating how the drugs are delivered. Drug delivery technology spansover all forms of administration from oral to injectable to implants toskin patches. Most of these technologies make use of an encapsulationtechnique or bead technology wherein the active ingredient isencapsulated or “trapped” inside a polymeric sphere. This polymericsphere can exist as a micelle, as a self assembled molecular rod or ballor a coating around the active ingredient. The drug is released bysolvation or swelling from the encapsulating agent as it circulatesthrough the blood or traverses the gut. The main advantage of modulatingthe delivery of the drug is to extend its release, modulate the bloodlevels for improved safety or enhance its absorption for improvedefficacy. Thus it is an embodiment of this invention that drug-metalcomplex release is modulated in vivo by physicochemical action on thecomplex itself.

In certain cases it may be beneficial to enhance the stability of theactive agent-metal complex by encapsulating the drug-metal complexwithin a porphine, peptide or polymeric matrix. This is particularlytrue if the active agent does not contain the necessary elements forforming a stable complex with a metal, such as with the primary aminesor alcohols mentioned above. It is a preferred embodiment of theinvention that the matrix be a porphine derivative, modified, ifnecessary, to allow bonding of the active agent to the metal. It is anembodiment of this invention that drug-metal complex release ismodulated in vivo by physicochemical action on the porphine, peptide orpolymeric matrix. It is a further preferred embodiment of this inventionthat the matrix be a compound found naturally in the small intestines.In yet a further preferred embodiment of this invention the porphinematrix is bilirubin or a derivative thereof.

It is a further preferred embodiment of the invention that theencapsulating matrix is an amino acid or dipeptide, wherein amino acidsor multiple dipeptides can be added to coordinate with or self assembleabout the metal-ligand complex. Histidine is an ideal amino acid due tothe strong metal binding capacity of the imidazole moiety in histidine.Arginine is another amino acid well suited for complexation withmagnesium through amidinate ligation of the guanidine portion of peptidebound arginine (FIG. 8). In a related embodiment of the invention,magnesium, due to its complexing and acid neutralizing, would stabilizearginine in the stomach and increase it potency. This is good for whenarginine is used as a NO source to help with COPD and related diseasestates.

The use of amino acids as secondary ligands on the metal is to stabilizethe inner coordination sphere, create a hydrophobic shell about theinner sphere and thus preventing hydrolysis of the metal-drug bond.Thus, it is an embodiment of the invention that amino acids, dipeptidesor oligopeptides act as secondary ligands or adjuvants on the metal-drugcomplex to stabilize the complex, particularly in aqueous systems. It isa preferred embodiment of the invention that the secondary ligand is adipeptide. It is another preferred embodiment of the invention that thesecondary ligand is an amino acid. In yet another preferred embodimentof the invention the amino acid is selected from the group histidine andarginine.

Organometallic complexes that have a free amino group (e.g. having anamino acid as part of the complex such as histidine) can initiatepolymerization of an amino acid-NCA to form a polypeptide,conformationally protecting the organometallic complex. It is a furtheradvantage of this technique to allow the amino acid NCA's to selfassemble about the organometallic complex and then coacervating thepolypeptide into its self assembled structure upon initiation ofpolymerization.

It is an embodiment of this invention to combine the encapsulationtechnology with the covalent technology to form an inner sphere covalentbond between the active agent and the transition metal, thus making anew composition of matter, and then encapsulating the complex with outersphere coordination within a polymer matrix to provide a stable complex.FIG. 9 illustrates an active agent (for structural simplicity salicylicacid is the example used), and polymer bound arginine bond to magnesiumin the inner sphere and peptides encapsulating the complex in the outersphere.

It is a further embodiment of the invention that the active agent onlybe released when the encapsulating matrix swells or is dissolved bywater, oil, emulsions or biologic fluids such as gastric juices. It isan embodiment of the invention that the active agent cannot be releasedfrom the encapsulating matrix by virtue of the strong bond between theencapsulating agent and the active agent, such as what would occur withan antibody-antigen complex. In some cases it would be beneficial tohave the release of the drug from the encapsulating agent be modulatedby digestive enzymes. It is a preferred embodiment of the invention thatthe active agent is released from the encapsulating agent by itschemical breakdown by enzymes secreted in the intestines, within thecell membrane or circulating in the blood stream. It is preferredembodiment of the invention that the active agent is bound to aluminum,magnesium, calcium, iron, bismuth, silicon or zinc. In another mostpreferred embodiment of the invention the encapsulating agent is anantibody raised against the metal-ligand complex. In yet anotherembodiment of the invention the complex comprises an active agent-metalcomplex and the encapsulating agent is self-assembled from thecombinations of amino acids, porphines, carbohydrates or mixturesthereof. In a most preferred embodiment of the invention the activeagent-metal-encapsulating agent complex is a pharmaceutical.

In another embodiment of the invention, the coordination complex is ametal selected from all metals that can form such complexes, and thedrug is selected from the group of all biologically or pharmacologicallyactive agents. In a preferred embodiment of the invention thepharmacologically active agent requires a specific conformation forbiological activity. The activity could be dependant on the activeagent's ability to cross cell membranes and the coordinating metalprovides the correct structure for membrane translocation of the activeagent. In a preferred embodiment the pharmaceutically active agent isselected from the group consisting of small molecules, peptides,carbohydrates, DNA or RNA, the latter two being used in gene therapy, asaptamers or in antisense nucleotide therapeutic applications. In apreferred embodiment the metal is selected from the group consisting ofaluminum, bismuth, calcium, magnesium, iron, silicon and zinc.

Bioadhesion Properties

There are a variety of ways that incorporating a magnesium or calciumion into the molecular formula of a drug would infer bioadhesiveproperties to the drug. For example, it is known that magnesium andcalcium are important for adhesive functions of integrins, thus it isreasonable to expect that a magnesium or calcium salt or complex of adrug in the intestinal tract would enhance bioadhesion of the drug tointegrins expressed on the brush border membrane of the intestinallining. And since bioadhesion translates into slower transit time in thegut, these complexes will confer sustained period of absorption in thegut. Therefore, it is an embodiment of this invention that sustainedabsorption of a drug will be enhanced by complexing the drug withmagnesium or calcium. It is a further embodiment of the invention thatsaid sustained release is conferred upon the magnesium or calcium drugcomplex by virtue of stronger bioadhesive properties.

Prevent Abuse of Narcotics

Narcotics are very effective analgesics but also can be very addictive.There have been many reports in the last few years describing the abuseof OxyContin by opiate addicts and recreational drug users. Typicallythe drug abuser will break the tablet matrix down mechanically orchemically, by adding water for example, thus making the full 12 hourdose available all at once. In addition, this type of abuse, whichusually starts with oral administration, can often lead the abuser tosnort or inject the concentrated narcotic.

Analysis of the structure of oxymorphone and oxycodone reveals that themolecules are ideal candidates for chelation with a metal. Theβ-hydroxyl at the 9-position and the nitrogen are positioned in such away that complexing a metal between the two would form a highlythermodynamically favored 5-member ring. It is preferred that the9-hydroxyl is deprotonated to form an anionic alkoxide (FIG. 10). Thenitrogen's lone pair of electrons may contribute enough electron densityto stabilize the metal chelate. Further stabilization can be imparted byadding secondary ligands or adjuvants to the complex in the manner ofthe case where salicylaldehyde stabilizes the glycine-magnesium complex.It is a further embodiment of the invention that the metal-narcoticcomplex is encapsulated within the matrix as described above.

Thus it is an embodiment of the invention that by virtue of the narcoticbeing complexed with a metal that the narcotic is released from thecomplex slowly through physicochemical action. This means that thenarcotic is not available immediately or all at once. Furthermore, it isan embodiment of this invention that the metal-narcotic complex isunable to pass the blood brain barrier, rendering the narcoticineffective until release from the complex has occurred. Since thekinetics of release is slow the amount of narcotic available fortransport across the blood brain barrier at any one time is much lessthan the dose administered and so no euphoric effect is achieved. It isa further embodiment of the invention that the kinetics of narcoticrelease can be slowed even more by incorporating secondary ligands,encapsulating agents or a combination of both. In a preferred embodimentthe metal is selected from the group consisting of aluminum, bismuth,calcium, magnesium, iron, silicon and zinc. In a more preferredembodiment of the invention the metal is selected from the main groupelements. In an even more preferred embodiment of the invention themetal is selected from the s-block main group elements. In a mostpreferred embodiment of the invention the metal is magnesium.

Selection of Metals

Reference has been made to the preferred metals to be used in thecoordination complexes. In pharmaceutical applications, the safety ofthe entire metal coordinated pharmaceutical needs to be considered whenselecting the metal used in complexes of this invention. Although topractice this invention many metals can be used, it is a preferredembodiment of this invention that the metal be selected from a shortlist that would be generally regarded as safe (GRAS). One criterion forselecting the metal is to review the list of mineral supplementscurrently on the market and select the ones whose dosages would farexceed the dose likely to be included as a coordination complex with thedrugs listed in Table 1. From the PDR for Non-prescription Drugs andDietary Supplements a list of 7 metals (excluding alkaline metals, i.e.sodium, potassium, etc) with amounts greater than 2 mg/dose is shown inTable 2.

TABLE 2 Coordination complex candidates from the PDR forNon-prescription Drugs Metal Compound Brand Name Amt. metal/doseAluminum Aluminum Hydroxide Maalox 400 mg Bismuth Bismuth SubsalicylatePepto-Bismol 525 mg Calcium Calcium Carbonate Caltrate 600 mg IronFerrous Fumarate Ferretts 106 mg Magnesium Magnesium Hydroxide Maalox400 mg Silicon Sodium Metasilicate One-A-Day  6 mg Zinc Zinc OxideOne-A-Day  15 mg

In a preferred embodiment of the invention the metal is selected fromthe group consisting of aluminum, bismuth, calcium, iron, magnesium,silicon and zinc. Whereas it is the embodiment of the invention that anew composition of matter is formed through the formation of a covalentbond between a pharmaceutical and any metal, including the lanthanides,actinides, the transition metals, and the main group metals (s- andp-block), it is a preferred embodiment of the invention that the metalbe selected from the s-block main group elements. The reason for this isthat the s-block elements are more likely to be GRAS and are more oftenused in OTC drug products and vitamin supplements than the transitionmetals or p-block main group elements (lanthanides or actinides arenever used in OTC products). There are several reasons for selectingmagnesium over the other s-block elements, such as calcium, which are:

-   -   1. Calcium shows larger variability with respect to coordination        number with 8>7>6>9 in order of preference. Magnesium, being        smaller than calcium, is almost exclusively octahedral, which        simplifies the synthetic strategies and will more likely give a        higher yield of a single product instead of a mixture of        products.    -   2. Magnesium can form covalent bonds with chelating ligands more        readily than the other s-block elements;    -   3. Magnesium forms a more stable bond with proteins and nucleic        acids than calcium and thus provides enhanced stabilization of        biologic pharmaceuticals.    -   4. Magnesium deficiency has been implicated in several disease        states (e.g., cardiovascular related, migraine headaches, ADHD),        and so from a prophylactic point of view magnesium may have        significant benefit. For example, triptan magnesium may be an        ideal candidate for this technology.    -   5. Calcium is absorbed in the intestines by an active transport        mechanism, whereas magnesium is transported passively. Magnesium        (as a salt) and Furosemide's intestinal transport were        facilitated when both were co-administered orally. Thus        Furosemide, a poorly absorbed drug, represents another        compelling candidate for this technology.

It is a most preferred embodiment of this invention that the metal ismagnesium.

Selection of Solvents

As stated earlier, the selection of solvent for the complexationreaction has an impact on the structure and stability of the metalcoordinated compound. Magnesium forms strong bonds with water and thecoordination sphere hydrated magnesium will have an impact on thekinetics of product formation as well as the structure and stability ofthe product. Because of the strong nitrogen-transition metal bond, inthose cases where nitrogen containing ligands are reacted withtransition metals, such as zinc, the presence of water in the reactionmixture will usually not have as strong an impact on the structure andstability of the metal coordinated product.

In some cases, depending on the ligand, the metal and the desiredproduct water may be the solvent of choice. The majority of the productswill dictate that an anhydrous organic solvent will be the bestselection. Some suitable solvents include alcohol, acetone or THF. Themost preferable solvent is DMSO because it is an excellent universalsolvent that dissolves virtually every pharmaceutical or nutraceuticaland also will dissolve most metal halides including magnesium chloride.This allows for single phase reactions. In addition, a stable metalcoordinated pharmaceutical can be isolated by a process similar tocoacervation, which typically will include simply adding a non-solventto the reaction mixture.

DMSO can form complexes with metals, including magnesium, in situ,setting up the DMSO-metal complex to react with the drug ligand, therebydisplacing the DMSO ligand at the metal center. DMSO can then serve as atransient protecting group in those reactions where adjuvants are to beincluded in the complex. This in-process reaction scheme is facilitatedby the fact that the DMSO-metal complex cannot form outer coordinationspheres like water does due to the lack of hydrogen bonding between theDMSO ligands. This makes the metal center easily accessible by incomingligands. Depending on the metal coordinated complex formed, the finalproduct may or may not retain DMSO as a ligand. If DMSO is attached tothe ligand, it is unlikely that a situation will exist such that thedosing of DMSO will ever reach anywhere close to toxic levels.

It is the premise of this invention that by merely adding a metal saltto an aqueous solution of a biologically active ligand the coordinationcomplex formed is not the same as if the combination of the reagentswere done under anhydrous conditions. Furthermore, by reconstituting thedried coordination complexes in the same aqueous environment thestructure of the two complexes would be different. To that end, severalcoordination complexes can be prepared with FDA approvedpharmaceuticals, demonstrating that the complexes are stable. Theproducts will be characterized as completely as possible andbioavailability studies will be conducted. The metal coordinatedcomplexes can be prepared in water and organic solvents. It is expectedthat, in many cases, the respective products will have differences instability, structure or biological activity.

Selection of Drugs

Complexes of almost any drug that can form a stable complex with a metalis enabled by this invention. The drugs selected for examples belowrepresent a cross section of chemical and therapeutic classes as shownin Table 2.

TABLE 3 Drugs selected as examples in the invention Drug TherapeuticClass Chemical Class Triidothyronine Hypothyroid drug Amino AcidMinocycline Antibiotic β-diketone Tetracycline Antibiotic β-diketoneHydrochlorothiazide Diuretic Sulfonamide Metformin Diabetes drugBiguanide Acycloguanosine Antiviral Ureide iRNA Gene therapyOligonucleotide

Small Molecule Discussions Synthesis

Other drug-magnesium complexes that may be important include Triptan-Mgdue to the importance of magnesium for headache relief and Oxycodone-Mgbecause of the importance of abuse resistant narcotics.

Typically an anhydrous metal halide (iodide, bromide or chloride) isadded to a mixture of the drug and KO^(t)Bu in ^(t)BuOH/DMSO.Alternatively, the metal halide can be added to the drug plus a solutionof a tertiary amine (e.g. triethylamine) in DMSO. Yet another option iswhere the metal halide can be added to the drug plus KH in THF. Themetals of choice are magnesium and zinc and the halide of choice ischloride. Zinc chloride is soluble in DMSO, acetone or ethanol and arethe solvents of choice for zinc complexation, particularly with nitrogencontaining ligands.

The product is isolated by precipitation, is separated from the liquidby suction filtration or centrifugation, washed and then dried underhigh vacuum to remove the last traces of moisture. The drug:metalcomplex may form a hydrate and all of the water may not be removableunder high vacuum. Alternatively, the added water may not displace theremaining DMSO ligands on the metal formed in situ. Consequently, theproduct may be a drug:metal:DMSO complex.

Certain drugs, where their dissociation constants are high when bound tomagnesium, favor complexation with DMSO. Formation of ternary complexesin situ would further stabilize the complexes and would retain theirmolecular integrity during the process of absorption after oraladministration. It is for this reason that for most drugs when reactedwith magnesium halide, DMSO is the solvent of choice.

As a comparator for the complexation reactions in the examples(excluding the acycloguanosine-Mg and T3-Zn examples), the reaction isrepeated except water is included in the reaction medium as described inthe examples below. The reaction is worked up and dried as before.

Adding water to the T3-Mg complexation reaction clearly had an impact onthe isolated product. The T3-Mg compounds prepared in DMSO alone, showedline broadening in the aliphatic region only, with sharp aromatic peaks,in its ¹H NMR spectrum (FIG. 11).

The ¹H NMR of T3, which shows the sharp peaks in the aliphatic region,is shown in FIG. 12. By comparison, the ¹H NMR of the T3-Mg productprepared in the presence of water revealed extensive line broadeningthroughout the ¹H NMR spectra. Furthermore, the magnesium content(1.62%) of the anhydrously prepared T3-Mg product very closely matchedthat of bis(triiodothyroninato)-bis(dimethylsulfoxide)-magnesium. Incontrast, the T3-Mg complex prepared in the presence of water had amagnesium content of only 0.96%. It also had 0.23% potassium in it,whereas no potassium was detected in thebis(triiodothyroninato)-bis(dimethylsulfoxide)-magnesium product.

Tetracycline has β-diketone and β-ketophenol functionalities and willform stable complexes with magnesium. Adding water to the reaction inDMSO has very little effect on the solubility, the ¹H NMR spectra or themetal content of the respective products. In fact the ¹H NMR spectrum ofthe product isolated from a reaction done in water alone does not differsignificantly from the product isolated from DMSO alone or in a 5:1DMSO:water mixture. There is a trend of lower magnesium content withhigher water content in the reaction solvent, but that may be due,primarily, to extent of hydration in the product.

Since zinc forms a very stable bond with nitrogen containing compounds,water does not interfere with complexation between zinc and the ligandbut may impact the complex structure. The product resulting fromreacting a metal and a drug in anhydrous DMSO typically yielded wellcharacterized coordination complexes. For those compounds that wereeither not well defined structurally (i.e. HCTZ-Zinc) or were somewhatunstable (i.e., dimethylbiguanide-zinc complex), a zinc coordinationcomplex was isolable and was, at least partially, characterized. Thesame complexes prepared in the presence of water, had higher solubilityin polar solvents. The difference in solubilities of the products fromthe respective methods of preparation clearly established a differencein the products themselves. It is believed that the zinc productsprepared in aqueous solvent systems produced ionic salts, outercoordination complexes, hydrated complexes or combinations thereof. Thisappeared to be the case in the dimethylbiguanide-zinc complex preparedin 5:1 DMSO:water mixture, where the ¹H NMR spectrum revealed resonancesat 2.85 and 2.80 ppm, which correspond to the N-methyl groups andindicate free (or ionic) and complexed dimethylbiguanide, respectively.

Characterization

Each product synthesized were characterized by NMR, MS (either TOF orFAB) and ICP. The NMR spectrum confirms the integrity of the sample andshows that a metal is complexed by the presence of line broadening, peakshifts or multiple resonances.

Metal content for most of the complexes prepared in anhydrous DMSO wereconsistent with a complex of two drugs bound to a metal. In addition,the metal content remained constant from batch to batch. The complexesof Dimethylbiguanide had variable metal content depending on the methodof isolation and never had consistent drug:metal ratios. The metalcontent was determined by the ICP analysis and based on that data, inconjunction with the NMR and MS, the ratio of drug to metal can becalculated.

The ¹H NMR spectra of the T3 complexes showed some line broadening andupfield shifting in the aliphatic region indicative of complex formationwith the amino acid portion of the molecule. This can be seen bycomparing the region between 2.5 ppm and 3.5 ppm in the ¹H NMR spectraof, bis(T3)bis(DMSO)Mg, T3, free acid and bis(T3)Zn, which are shown inFIGS. 11, 12 and 13, respectively.

The ¹H NMR spectrum of the dimethylbiguanide complexes showed largeupfield shifts of the —NH resonances indicative of complex formationwith the nitrogen atoms. In addition, a 0.05 ppm upfield shift ofN-dimethyl groups was observed in the dimethylbiguanide-zinc complexspectrum (FIG. 14) relative to the spectrum of dimethylbiguanide (FIG.15)

The ¹H NMR spectrum of the minocycline and tetracycline complexesresembled polymeric structures with very large line broadening andmanifestation of many new resonances throughout the entire spectrum. Todemonstrate that these spectra were due to dynamic isomeric mixtures andnot decomposition or polymerization, 12 N HCl was added to the NMRsamples of tetracycline and its magnesium complex and the spectraretaken. FIGS. 16-18 show the ¹H NMR spectra of Tetracycline,bis(tetracyclinato) magnesium and the complex with HCl added,respectively. As can be seen in the spectra series the magnesium complexreverted back to the reference tetracycline compound. Interestingly,when HCl was added to tetracycline, a considerable amount ofdecomposition could be seen in the aromatic region of the NMR spectrum(FIG. 19), the magnitude of which was not seen in the commensuratespectra of the tetracycline-magnesium complex. This indicates an acidprotective effect imparted by complexation with magnesium and couldbecome an important attribute of this technology for those drugs thatare unstable in acid environments, such as in the stomach.

The ¹H NMR spectrum of the hydrochlorothiazide complexes also resembledpolymeric structures with the same kind of line broadening and newnondescript resonances seen in the spectra of the antibiotic-metalcomplexes. FIGS. 20-22 show the ¹H NMR spectrum of hydrochlorothiazide,hydrochlorothiazide-zinc complex and the complex with HCl added,respectively. As can be seen the line broadening was reverted back tothe sharp resonances observed in the reference drug. This proves thatthe line broadening and additional resonances observed in the ¹H NMRspectrum of the respective complexes were due to multiple stereochemicaland geometric isomers of the complex in solution. Moderately slowinterchange between the isomers in solution could also contribute to theline broadening observed.

Spectral data on the acycloguanosine-magnesium complex data showed thata complex was formed. Comparison of the ¹H NMR spectra ofacycloguanosine with that of its magnesium complex (FIG. 23) suggestedthat the complexation site on acycloguanosine was the amide oxygen andthe imidazole nitrogen; the resonance at 10.6 ppm, which is missing inthe NMR spectrum of the complex, is assigned to the amide proton.

The mass spectrum revealing a significant presence of the coordinationcomplex is an important indicator of the stability of the complex. Thus,molecular ions were found for bis(T3)Mg, bis(T3)Zn,bis(minocyclinato)Mg, bis(tetracyclinato)Mg, andbis(acycloguanosinato)Mg. Two molecular ions with zinc isotope patternswere observed in the MALDI spectrum of hydrochlorothiazide-zinc complex.It is not known, at this time, a structure corresponding to thosemasses. Dimethylbiguanide-zinc complex did not have a zinc containingmolecular ion in its MALDI spectrum. This is believed to be due to thecompound's instability.

FTIR studies may be used to determine whether, for a particular complexthat has been found, the ligand bonding atom and if the complex iscoordinated with DMSO, water, or not solvated at all.

Stability

Equilibrium constants for the coordination complexes made have beenestimated from literature precedents of similar compounds. For example,the equilibrium constant, log K_(eq) for T3-Zn is estimated to bebetween 4 and 5 based on another amino acid zinc complex,phenylalanine-zinc. Likewise, the log K_(eq) for dimethylbiguanide-zincis estimated to be between 5 and 7. The log K_(eq) forhydrochlorothiazide-zinc is difficult to estimate from literaturevalues. Stability constants for tetracycline-magnesium in water atvarious pH values have been reported and the expected log K_(eq) fortetracycline-magnesium is between 4 and 5. The log K_(eq) foracycloguanosine-magnesium is estimated to be 1.6. The log K_(eq) forT3-Mg is difficult to estimate due to the lack of a good comparator butthe log K_(eq) for glycine, which like T3 is also an amino acid, is1.34. Due to T3's much greater hydrophobicity relative to glycine, thelog K_(eq) for T3-Mg is expected to be much larger than 1.34.

Another indicator of drug-metal or drug-metal-adjuvant stability istheir binding constants, which is related to K_(eq) but further showsthe stepwise stability for multidentate ligands. The cumulative bindingconstant, β_(n), for the maximum binding between the metal and theligand is given by Equation 4.

$\begin{matrix}{\beta_{n} = {\frac{\left\lbrack {ML}_{n} \right\rbrack}{{\lbrack M\rbrack \lbrack L\rbrack}^{n}} = {\sum\limits_{i = 1}^{n}K_{i}}}} & {{Equation}\mspace{14mu} 4}\end{matrix}$

This difference can be seen in the measured log K_(eq) forphenylalanine-zinc and its approximated β₂ value of 8.5. This β₂ valuemay also more closely reflect the stability of the T3-Zn complex.

There are several ways in which binding constants of metal-drugcomplexes in different environments can be estimated. Optical absorptionspectroscopy, NMR spectroscopy, mass spectrometry, reaction kinetics,potentiometry and chromatography are several such methods.

Partition Coefficient and Distribution Coefficient

The partition coefficient is a constant and is defined as the ratio ofconcentration of a neutral compound in aqueous phase to theconcentration in an immiscible organic phase, as shown in Equation 5.

Partition Coefficient,P=[Organic]/[Aqueous]  Equation 5

In practice the Log P, defined in Equation 6, will vary according to theconditions under which it is measured especially pH since at a low pHbases will be ionized and at a high pH acids will be ionized.

Log P=log 10 (Partition Coefficient)  Equation 6

Thus, a Log P=1 means 10:1 Organic:Aqueous, a Log P=0 means 1:1Organic:Aqueous and a Log P=−1 means 1:10 Organic:Aqueous.

Naturally, ionized compounds will partition preferentially into theaqueous phase, thereby lowering their log P. For neutral molecules thatare bases they will remain neutral when the pH is greater than 2 unitsabove its pKa and for neutral acids when the pH is 2 units below itspKa.

The choice of partitioning solvent will also have an impact on log P.Most log P measurements will use the octanol:water system. Ion pairingeffects impact the log P measurements and should be accounted for,especially with metal coordinated compounds such as those embodied inthis invention.

In terms of pharmaceutical applications the following guidelines havebeen used in determining the method of administration, formulation anddosage forms:

-   -   Low Log P (below 0) Injectable    -   Medium (0-3) Oral    -   High (3-4) Transdermal    -   Very High (4-7) Toxic build up in fatty tissues

And within the realm of orally administered drugs these guidelines havebeen used:

-   -   1. For optimum CNS penetration, Log P=2+/−0.7 (Hansch rules)    -   2. For optimum oral absorption, Log P=1.8    -   3. For optimum intestinal absorption, Log P=1.35    -   4. For optimum colonic absorption, Log P=1.32    -   5. For optimum sub lingual absorption, Log P=5.5    -   6. For optimum Percutaneous penetration, Log P=2.6 (& low mw)

The distribution coefficient (D) is the ratio of unionized compound inthe organic phase to the total amount of compound in the aqueous phasegiven by Equation 7.

D=[Unionised](o)/[Unionised](aq)+[Ionised](aq)  Equation 7

Log D is the log distribution coefficient at a particular pH (Equation8). This is not constant and will vary according to the protogenicnature of the molecule. Log D at pH 7.4 is often quoted to give anindication of the lipophilicity of a drug at the pH of blood plasma.

Log D=log₁₀ (Distribution Coefficient)  Equation 8

Log D is related to Log P and the pKa by the following equations:

Log D _((pH))=log P−log [1+10^((pH-pka))] for acids  Equation 9

Log D _((pH))=log P−log [1+10^((pKa-pH))] for bases  Equation 10

So, when the pH is adjusted such that ionization is minimized, the log Dwill be nearly equivalent to the log P. Under those conditions, then,log D is a reliable indicator of the bioavailability of a drug in aparticular application. In terms of a metal coordinated drug, increasesin log D of the drug-metal complex relative to the reference drug willnot only indicate an increase in lipophilicity but will also demonstrateits stability in water, as well. The log D's at pH 7.4 for tetracycline,bis(tetracyclinato)magnesium, triiodothyronine andbis(triiodothyroninato)zinc were determined and are shown in Table 4along with their pKa's.

TABLE 4 pKa and Log D for selected reference drugs and their metalcoordination complexes. Compound Method pKa Log D_(7.4) TetracyclineDPAS 8.95, 7.04, 3.34 −1.2 Bis(tetracyclinato)magnesium DPAS 8.33, 7.17,3.36 0.01 Triiodothyronine Potentio- 8.41, 8.07 2.80 metricBis(triiodothyroninato)zinc Potentio- 6.66, 6.42 3.43 metric DPAS = DipProbe Absorption Spectroscopy

These results run counter to what the prior art teaches; that iscombining the anion of a neutral compound with a metal salt shouldproduce a compound with less lipophilicity and a reduced log D. Perhapsmore interestingly, by application of the technology in this invention,tetracycline was theoretically transformed from an injectable drug (logD<0) to an oral drug (log D>0) and T3 was transformed from an oral drug(log D<3) to a transdermal drug (log D>3). This latter manifestation hasimportant implications in increasing the safety of T3 products byadministering the drug in a slow release transdermal depot.

Bioavailability Studies Overview:

A preliminary study in a rat model to observe the effects thatcoordinating a metal with a drug will have on the absorbance of thereference drug was conducted. The reference drug selected for thisparticularly study was triiodothyronine (T3), which is the activeingredient in Cytomel and Thyrolar. Both Cytomel and Thyrolar arecurrently used to treat hypothyroidism. Cytomel has also been indicatedin the treatment of certain psychological disorders.

Study Design:

T3-Zinc and T3-Magnesium were tested for bioavailability, relative tothe reference drug over a 5 hour time period. The three compounds wereformulated, separately, into gelatin capsules, with a total dose of108±12 μg/kg administered. In order to avoid acid degradation of thetest compounds in the rat stomach, metal oxides were added to theformulation. Another T3 control was included, where zinc oxide was addedto T3, free acid. Each of the formulated gelatin capsules were orallyadministered directly to the esophagus of respective rats and bloodsamples were collected at pre-dosing and at 0.5, 1, 2, 2.5, 5 hoursafter dosing. Serum triiodothyronine levels were analyzed by anindependent laboratory, using an industry standard assay method.

Results:

The data shown in the FIG. 30 reveal that all four drug formulationswere readily absorbed by the rat, with a rapid rise in serum T3 levelsup to the 2.5 hour time point and a general leveling off in the metalcoordinated T3 fed rats after that. The T3, free acid fed rats indicatedthat the serum T3 levels may still be rising at the 5 hour time point.The rats that were administered metal complexed T3 and T3 with zincoxide added had an increase in serum T3 blood levels that wereapproximately 65-85% greater that of T3, free acid at the 5 hour timepoint.

Conclusions:

The data clearly indicate that complexing a metal with T3 increased theabsorbance of orally administered T3 nearly two fold over T3 alone. Itis believed that the T3, free acid complexed with the zinc oxide priorto absorption of T3 in the animals fed the formulation of T3 and zincoxide.

This study represents a significant advance in drug delivery technologyand that coordinating metals with pharmaceuticals can be applied to andimprove the performance of drugs with bioavailability limitations.

Large Molecule Protocols Synthesis

Double stranded iRNA of a defined size were prepared according to thestandard protocol described in the Examples section. The iRNA was thenreacted with magnesium or zinc under anhydrous conditions or in thepresence of water as described in the Examples section.

The biological activity of iRNA can be modulated in various ways bycomplexing it with other metals such as calcium, zinc, cobalt andmanganese. In addition, combinations of multiple metals, such asincluding Cu or Ag³³ to facilitate binding of the purine/pyrimidinegroups along with the phosphate groups can improve the transfectionefficiency and stability of iRNA.

Characterization

The metal:RNA products were tested for changes in ionic/covalentbehavior using Isoelectric focusing (IEF) gel following the standardprotocol described in the Examples section. The advantage of the IEF gelis that it provides an inexpensive tool by which to monitor changes inthe charge distribution on the target RNA molecule. Data from the IEFstudies were represented as ΔpKa for each iRNA complex prepared.

Conclusion of IEF Experiments

The presence of an RNA molecule from the anhydrous DMSO reactions with adifferent isoelectric point (pKa) indicates the presence a new RNAmolecule (FIG. 31). Since this is not due to RNA degradation means thatthe new RNA molecule is a stable complex between the metal (magnesium orzinc) and RNA. In addition, because its pKa is lower than the native RNAsupports the formation of a covalent bond between the metal and RNA.

Examples FTIR Analysis of DMSO-Magnesium Complex

In order to determine which atom of DMSO binds to magnesium and FTIRspectrum was collected of a DMSO-magnesium complex. The FTIR spectrumshowed an extra stretch at 954 cm⁻¹, which is indicative of an S═O—Mgstretch. FTIR of T3 complexes were examined for the presence of S═O—Mg,C═O and N—H stretches.

Preparation of Bis(triiodothyroninato)-bis(dimethylsulfoxide)magnesium

Triiodothyronine or T3 (218 mg) was dissolved in 4 mL of anhydrous DMSO,after which 0.34 mL of 1 M potassium t-butoxide in t-butanol was addedand the solution stirred for 10 minutes. Magnesium chloride (16 mg) wasadded and the solution stirred overnight. The solution was poured into10 mL of deionized water to precipitate the product, which was suctionfiltered and air dried. After an overnight drying under high vacuum theyield was 164 mg of a light beige powder. The product structure wascharacterized by ¹H NMR, FAB-MS and ICP. ¹H NMR (DMSO): δ 7.83 (s), 7.05(d), 6.82 (d), 6.62 (dd), 3.26 (bm), 3.15 (bd), 2.71 (bm), 2.54 (s). Thepresence of the broad multiplets in the side chain region indicates thatas the site for magnesium binding (see FIG. 11). FAB-MS: Molecular ionat 1325 indicative of bis(triiodothyroninato)magnesium (after loss ofDMSO ligands). FTIR (neat): cm⁻¹ 1596 (C═O stretch), 1014 (S═O stretch),949 (S═O—Mg stretch); absence of N—H stretch at 1633. Magnesium analysis(ICP): Expected 1.68%. Found 1.62%. This confirms the structure as shownin FIG. 24 and is about 96% pure, where most of the contamination isprobably due to water as seen in the ¹H NMR spectrum.

Preparation of Bis(triiodothyroninato)zinc

Triiodothyronine or T3 (192 mg) was dissolved in 4 mL of anhydrous DMSO,after which 0.30 mL of 1 M potassium t-butoxide in t-butanol was addedand the solution stirred for 10 minutes. Zinc chloride in diethyl ether(0.16 mL of 1M solution) was added and the solution stirred overnight.The solution was poured into 10 mL of deionized water to precipitate theproduct, which was suction filtered and air dried. After an overnightdrying under high vacuum the yield was 140 mg of a light beige powder.The product structure was characterized by ¹H NMR, FAB-MS and ICP. NMR(DMSO): δ 7.82 (s), 7.02 (d), 6.80 (d), 6.61 (dd), 3.49 (bm), 3.22 (bm),2.67 (bm). The presence of the broad multiplets in the side chain regionindicates that as the site for zinc binding (FIG. 13). FAB-MS: Molecularion at 1364 and 1366 indicative of bis(triiodothyroninato)zinc withisotopic abundance pattern consistent with zinc. FTIR (neat): cm⁻¹ 1582(C═O stretch); absence of N—H stretch at 1633. Zinc analysis (ICP):Expected 4.8%. Found 4.3%. This supports the structure as shown in FIG.25 and is about 90% pure, where most of the contamination is probablydue to water and may be a hydrate as seen in the ¹H NMR spectrum.

Preparation of Magnesium Triiodothyronine Complex in the Presence ofWater

Triiodothyronine or T3 (188 mg) was dissolved in 3.5 mL of anhydrousDMSO, after which 0.29 mL of 1 M potassium t-butoxide in t-butanol wasadded and the solution stirred for 10 minutes. Magnesium chloride (16mg) in 0.5 mL of water was added and the solution stirred overnight. Thesolution was poured into 10 mL of deionized water to precipitate theproduct, which was suction filtered and air dried. After an overnightdrying under high vacuum the yield was 188 mg of a light beige powder.The product structure was characterized by ¹H NMR, FAB-MS and ICP. ¹HNMR (DMSO): δ 7.81 (bs), 7.02 (bs), 6.81 (bd), 6.59 (dd). The otherresonances were hiding behind the solvent and water peaks. The sampleformed a cloudy suspension in DMSO. FAB-MS: Molecular ion at 652indicative of protonated T3 with no bound Mg. FTIR (neat): cm⁻¹ 1633(N—H stretch), 1535 (C═O stretch), 1012 (S═O stretch), 949 (S═O—Mgstretch). The strength of the DMSO related stretches relative to the T3related stretches indicated a mixture of (DMSO)_(x)Mg cation and T3anion. Magnesium analysis (ICP): Expected 1.8%. Found 0.96%. Potassiumanalysis (ICP). Found 0.23%. From the ICP data it appears that thisproduct is a mixture of the magnesium salt, the potassium salt and thezwitterion.

Preparation of Bis(minocyclinato)magnesium

Minocycline (104 mg) was dissolved in 3 mL of anhydrous DMSO, afterwhich 0.44 mL of 1 M potassium t-butoxide in t-butanol was added and thesolution stirred for 10 minutes. Magnesium chloride (11 mg) was addedand the solution stirred overnight. The solution was poured into 10 mLof deionized water to precipitate the product, which was suctionfiltered and air dried. After an overnight drying under high vacuum theyield was 52 mg of a deep yellow powder. The product structure could notbe characterized by ¹H NMR, possibly due to the different permutationsof bidentate complex forms possible with anionic Minocycline andmagnesium. The product was characterized, then, by FAB-MS and ICP.FAB-MS: Molecular ion at 937.4 indicative ofbis(minocyclinato)magnesium. Magnesium analysis (ICP): Expected 2.68%.Found 2.61%. This confirms that there are two Minocycline molecules permagnesium atom, as represented by a likely structure shown in FIG. 26(see discussion for Tetracycline below). The product about 97% pure,which most of the contamination is probably due to water as seen in the¹H NMR spectrum.

Preparation of Bis(tetracyclinato)magnesium

Tetracycline (89 mg) was dissolved in 0.5 mL of anhydrous DMSO, afterwhich 0.2 mL of 1 M potassium t-butoxide in t-butanol was added and thesolution stirred for 10 minutes. Magnesium chloride (11 mg) was addedand the solution stirred for 3 hours. The solution was concentrated invacuo at 30° C., after which 0.5 mL of deionized water was added and themixture triturated and transferred to a 2 mL microcentrifuge tube. Theproduct was separated from the liquid by centrifuging at 8,000 rpm for 6minutes and the supernatant was decanted. The pellet was washed byadding 0.5 mL of water, vortex mixed, centrifuged and the supernatantdecanted. The washing procedure was repeated. After an overnight dryingunder high vacuum the yield was 72 mg of a deep yellow powder. The ¹HNMR spectrum (FIG. 20) resembled a polymeric structure, which containedbroad multiplets between 8.8 and 10.1 ppm, 6.4 and 7.8 ppm, 4.2 and 5.0ppm and 1.1 and 3.1 ppm. The product structure could not be accuratelycharacterized by ¹H NMR, possibly due to the different permutations ofbidentate complex forms possible with anionic Tetracycline and magnesiumand the moderately slow equilibrium between those isomeric complexforms. Confirmation was observed by adding approximately 1 equivalent of12 N HCl to the NMR sample and reanalyzing by ¹H NMR, which revealedreversion of the magnesium complex back to tetracycline and, presumably,magnesium chloride (FIG. 18). The product was further characterized byMALDI-ES and ICP. MALDI-ES: Molecular ion at 911.3 indicative ofbis(tetracyclinato)magnesium. Magnesium analysis (ICP): Expected 2.67%.Found 2.53%. FTIR does not indicate presence of a DMSO ligand. Thisindicates that there are two Tetracycline molecules per magnesium atom.According to NMR evidence from previously published studies oftetracycline-magnesium complexation in aqueous systems, a likelystructure for bis(tetracyclinato)magnesium is shown in FIG. 27. Theproduct about 95% pure, which most of the contamination is probably dueto solvents as seen in the ¹H NMR spectrum.

Preparation of Magnesium Tetracycline Complex in Water

Tetracycline (89 mg) was dissolved in 1.5 mL of water, after which 0.2mL of 1 M potassium t-butoxide in t-butanol was added and the solutionstirred for 10 minutes. Magnesium chloride (0.11 mL of 1M solution) wasadded and the solution stirred for 3 hours. The resultant precipitantwas separated from the water by centrifuging at 8,000 rpm for 6 minutesand the supernatant was decanted. The pellet was washed by adding 1 mLof water, vortex mixed, centrifuged and the supernatant decanted. Afteran overnight drying under high vacuum the yield was 65 mg of a deepyellow powder. The ¹H NMR spectrum very closely resembled the complexprepared in anhydrous DMSO. Magnesium analysis (ICP): Expected 2.67%.Found 2.42%. It appears that performing the complexation in water versusunder anhydrous conditions has a minor impact on the stability andstructure of the tetracycline-magnesium complex.

Preparation of Hydrochlorothiazide Zinc Complex

Hydrochlorothiazide (120 mg) was dissolved in 0.5 mL of anhydrous DMSO,after which 0.4 mL of 1 M potassium t-butoxide in t-butanol was addedand the solution stirred for 10 minutes. Zinc chloride in diethyl ether(0.2 mL of 1M solution) was added and the solution stirred for 4 hours.The solution was concentrated in vacuo at 30° C., after which 0.5 mL ofmethanol was added and the mixture triturated and transferred to a 2 mLmicrocentrifuge tube. The product was separated from the liquid bycentrifuging at 8,000 rpm for 6 minutes and the supernatant wasdecanted. The pellet was washed by adding 0.5 mL of methanol, vortexmixed, centrifuged and the supernatant decanted. The washing procedurewas repeated. After an overnight drying under high vacuum the yield was104 mg of a free flowing white powder. The product was apparentlyhygroscopic due to the powder turning gummy after a few minutes exposureto ambient air. The ¹H NMR spectrum resembled a polymeric structure(FIG. 21), which contained broad multiplets between 6.3 and 8.0 ppm, and4.4 and 5.9 ppm. The product structure could not be accuratelycharacterized by ¹H NMR, possibly due to the different permutations ofcomplex forms possible with anionic hydrochlorothiazide and zinc, andthe moderately slow equilibrium between those isomeric complex forms.Confirmation was observed by adding approximately 1 equivalent of 12 NHCl to the NMR sample and reanalyzing by ¹H NMR, which revealedreversion of the zinc complex back to hydrochlorothiazide and,presumably, zinc chloride (FIG. 22). The product was furthercharacterized by MALDI-ES and ICP. MALDI-ES: Molecular ions at 705.9 and932.9 with typical zinc isotopic abundances but not indicative of anyparticular hydrochlorothiazide zinc complex structure. Zinc analysis(ICP). Found 10.4%. FTIR (neat): cm⁻¹ 1027 (S═O stretch), 953 (S═O—Znstretch); absence of N—H stretch at 1646. NMR, MAL DI and ICP dataclearly indicate the formation of a hydrochlorothiazide-zinc complex. Itseems reasonable that the site of complexation may be on one or both ofthe sulfonamide nitrogens of hydrochlorothiazide. FTIR data suggest thepresence of DMSO ligands, which by ¹H NMR integration the ratio ofHCTZ:DMSO is 1:1. Chemical shift of the DMSO methyl groups of 0.08 ppmsuggests O-bonding between zinc and DMSO.

Reaction Between Zinc and Hydrochlorothiazide in the Presence of Water

This procedure followed the analogous anhydrous preparation exactlyexcept the zinc chloride was added to 100 μl of water and the ether wasallowed to evaporate off prior to adding to the reaction mixture. Noproduct was isolated because the entire mixture slowly dissolved inmethanol during the precipitation step. This was putatively due to theformation of methanol soluble hydrated complexes.

Preparation of Dimethylbiguanide Zinc Complex

Dimethylbiguanide (66 mg) was dissolved in 1 mL of anhydrous DMSO, afterwhich 0.88 mL of 1 M potassium t-butoxide in t-butanol was added and thesolution stirred for 10 minutes. Zinc chloride in diethyl ether (0.22 mLof 1M solution) was added and the solution stirred for 3 hours. Thesolution was concentrated in vacuo at 35° C., after which 0.5 mL ofethanol was added and the mixture triturated and transferred to a 2 mLmicrocentrifuge tube. The product was separated from the liquid bycentrifuging at 8,000 rpm for 6 minutes and the supernatant wasdecanted. The pellet was washed by adding 0.5 mL of ethanol, vortexmixed, centrifuged and the supernatant decanted. The washing procedurewas repeated. After an overnight drying under high vacuum the yield was57 mg of a free flowing white powder. ¹H NMR (DMSO): δ 4.90 (s), 4.66(s), 4.50 (s), 2.80 (s). The large upfield shifts of —NH protonsrelative to dimethylbiguanide indicate complexation with zinc. A smallupfield shift of 0.05 ppm in the dimethyl groups was also observed inthe dimethylbiguanide-zinc complex. FIGS. 14 and 15 show the NMR spectraof dimethylbiguanide-zinc and dimethylbiguanide, respectively. Theproduct was further characterized by ICP. Zinc analysis (ICP). Found8.14%. FTIR data did not indicate presence of a DMSO ligand. NMR and ICPdata clearly indicate the formation of a dimethylbiguanide-zinc complex.FIG. 28 represents the biguanide-metal complex prepared. MALDI-ESanalysis did not reveal a zinc containing compound.

Preparation of Zinc Dimethylbiguanide Complex in the Presence of Water

This procedure followed the analogous anhydrous preparation exactlyexcept the zinc chloride was added to 100 μL of water and the ether wasallowed to evaporate prior to adding to the reaction mixture. Work upand drying was followed in the same manner to yield 47 mg of a freeflowing white powder. The compound was sparingly soluble in DMSOresulting in low signal to noise ratio in the NMR. ¹H NMR (DMSO): δ 4.90(bs), 4.66 (bs), 4.50 (bs), 2.85 (s), 2.80 (s). The singlet at 2.85 ppmindicates the presence of dimethylbiguanide that is not complexed withzinc. Judging from the integration of the two peaks at 2.85 and 2.80ppm, the ratio of free dimethylbiguanide to the zinc complex is about1:1.

Preparation of Zinc Dim ethylbiguanide Complex in Water

Dimethylbiguanide (33 mg) was dissolved in 1 mL of water, after which0.44 mL of 1 M potassium t-butoxide in t-butanol was added and thesolution stirred for 10 minutes. Zinc chloride in diethyl ether (0.22 mLof 1M solution) was added and the solution stirred for 5 hours. Theresultant precipitant was separated from the liquid by centrifuging at8,000 rpm for 6 minutes and the supernatant was decanted. The pellet waswashed by adding 1 mL of water, vortex mixed, centrifuged and thesupernatant decanted. After an overnight drying under high vacuum theyield was 20 mg of a free flowing white powder which contained noorganic material by ¹H NMR. The isolated product was zinc salts invarious hydrated forms.

Preparation of Bis(acycloguanosinato)magnesium

Acycloguanosine (45 mg) was dissolved in 0.5 mL of anhydrous DMSO, afterwhich 0.2 mL of 1 M potassium t-butoxide in t-butanol was added and thesolution stirred for 10 minutes. Magnesium chloride (11 mg) was addedand the solution stirred for 4 hours. The solution was concentrated invacuo at 30° C., after which 0.5 mL of methanol was added and themixture triturated and transferred to a 2 mL microcentrifuge tube. Theproduct was separated from the liquid by centrifuging at 8,000 rpm for 6minutes and the supernatant was decanted. The pellet was washed byadding 0.5 mL of methanol, vortex mixed, centrifuged and the supernatantdecanted. The washing procedure was repeated. After an overnight dryingunder high vacuum the yield was 15 mg of a beige coarse powder. Theproduct structure was characterized by ¹H NMR, MALDI-ES and ICP. ¹H NMR(DMSO): δ 7.70 (s), 6.75 (bs), 5.30 (s), 4.64 (bs), 3.42 (s). MALDI-ES:Molecular ion at 473.1 is suggestive of bis(acycloguanosine)magnesium.Other molecular ions at 666, 702, 893 and 1065 have not been assigned toa particular structure. Magnesium analysis (ICP): Expected 5.14%. Found4.16%. FTIR data did not indicate presence of a DMSO ligand. From acombination of the absence of the amide proton at 10.6 ppm, the ICPanalysis and the MALDI-ES analysis, the structure forbis(acycloguanosinato)magnesium is shown in FIG. 29.

Partition Coefficients, Distribution Coefficients and pK_(a) for T3 andTetracycline Complexes and Reference Drugs

Determination of pK_(a) and log P was done by potentiometry andspectrophotometry. The potentiometric method includes the use of expertsoftware to calculate pK_(a) and log P from simple acid and basetitrations of the analytes. The pK_(a) was first determined by weighingapproximately 2 mg of pure substance into an assay vial. Ionic strengthwas adjusted with 0.15M KCl and water was added to dissolve the compoundfollowed by an acid or base titrant to drop or raise the pH to thedesired starting value. The solution was then titrated with acid (0.5NHCl) or base (0.5N NaOH) to the final pH. Approximate pKa values weredisplayed and later refined to exact data.

For (T3)₂Zn, which is sparingly soluble in water, the pKa was determinedin mixtures of water and DMSO cosolvent. A minimum of three ratios ofwater/DMSO was titrated to obtain p_(s)K_(a) (apparent pK_(a) in thepresence of cosolvent). The aqueous pK_(a) was determined byextrapolation using the Yasuda-Shedlovsky technique.

The log P was determined by a titration in the presence of octanol(water saturated). The pK_(a) in water and the apparent pK_(a) in thepresence of octanol (p_(o)K_(a)) were compared and the log P determined.Ion-pairing (partitioning of a charged species into octanol, termed logP⁺ or log P⁻) were determined with an additional titration in thepresence of another volume of octanol. Using experimentally determinedpK_(a) and log P, a drug lipophilicity profile (log D vs. pH) wascalculated. The log D_(7.4) was determined from this profile at pH 7.4.

The spectrophotometric method used a fiber optics dip probe, a UV lightsource (pulsed deuterium lamp) and a photodiode array detector toautomatically capture the absorption spectra of the sample solution inthe course of adding an acid or base solution.

Up to a 10 mM stock solution was prepared by dissolving several 0.5 mgsamples in 0.5-1.0 mL of water or cosolvent. An adequate amount of stockis pipetted into the vial for titration. The dip probe is in the assayvial and aqueous 0.15 M KCl solution is added to cover the dip probe.Acid or base is added to bring the pH to the desired starting value.Over the chosen pH range, the spectra changes due to ionization werecaptured by the photodiode array detector for subsequent analysis.Target Factor Analysis (TFA) was applied to deduce the pK_(a) values ofthe sample and resolve the major absorptivity spectra of the reducingspecies. The aqueous pK_(a) was determined by extrapolation using theYasuda-Shedlovsky technique. The pK_(a) values obtained fromspectrophotometric experiments are in excellent agreement with thosederived from potentiometric titrations.

Bioavailability Study Title:

Assessment of absorbance and effect of a hormone complex supplementationin Sprague Dawley rats

Test Subjects:

Fifteen young female Sprague Dawley rats (180-225 gms) were used. Theserats were obtained from a commercial source (Harlan Laboratory Animals,Dublin, Va.), housed in the Vivarium at Litton Reeves Hall (Division ofLaboratory Animal Resources), in groups of three in polypropyleneshoebox cages. Water was available ad libitum. Rats were fed certifiedrodent chow ad libitum. After arrival, the health of rats were assessedand animals were placed in quarantine for a minimum of five days, duringwhich time, general health was assessed. At the end of quarantine, ratswere moved to permanent animal quarters for access and study.

Study Design:

This is a study to compare the absorption and effect of three hormonesadministered to rats orally directly into the esophagus. T3-Zinc andT3-Magnesium were synthesized, which are the two test compounds.Triiodothyronine, free acid (T3) was the positive control. The threecompounds were formulated, separately, into gelatin capsules, with atotal dose of 108±12 μg/kg administered. In order to avoid aciddegradation of the test compounds in the rat stomach, metal oxides wereadded to the formulation—to the T3-magnesium compounds, 1.08±0.13 mg ofmagnesium oxide was added and to the T3-zinc compounds, 106±13 μg ofzinc oxide was added. Another T3 control was included, where 145±50 μgof zinc oxide was added to T3, free acid.

For dosing and blood collection, three rats per replicate wereanesthetized. A baseline blood sample was collected, by retroorbitalsampling. Then, compound was administered orally by gavage tube.Following administration, blood samples (500 μL) were collected at 0.5,1, 2, 2.5, 5 hours after dosing. Blood samples were centrifuged, thesera removed and the sera kept on ice and then analyzed for serumtriiodothyronine levels.

Analysis of Samples:

Serum triiodothyronine levels were determined by RIA.

Results:

The individual serum T3 levels from each group of rats were averaged anda plot of T3 concentration (ng/mL) vs. hour was produced. The plot isshown in FIG. 30.

Large Molecule Example Preparation of Interference RNA

Interference RNA was prepared using a modified New England BiolabsLitmus 28i RNAi bidirectional transcription vector. A 922 bp bovineserum albumin cDNA fragment was introduced into the Bgl II and StuIsites of the Litmus RNAi vector. The target RNAi transcript was producedby in vitro transcription with T7 RNA Polymerase to yield 1 mg/ml. TheRNA was then divided into 50 μg samples and freeze dried

Preparation of RNA:Magnesium Inner Coordination (Covalent) Complex

Approximately 50 μg of iRNA was dissolved in 100 μL of anhydrous DMSO at50° C. A stock solution of 4 mM magnesium chloride was prepared bydissolving 19 mg of anhydrous magnesium chloride in anhydrous DMSO.Three separate reactions were run where 15 μL, 30 μL and 60 μL of 4 mMmagnesium chloride was added to three separate solutions of iRNA inDMSO. The solutions were allowed to set at room temperature withoccasional vortex mixing for 90 minutes at which time 20 μL of 7.5 Maqueous ammonium chloride followed by 400 μL of RNAse free ethanol wasadded and vortex mixed. The product was allowed to precipitate out ofsolution over 1 hour, centrifuged and the liquid decanted from thepellet. The pellet was washed with 100 μL of RNAse free ethanol, vortexmixed, centrifuged and the ethanol supernatant decanted off the pellet.The resultant colorless pellet was air-dried for several minutes beforetesting in the isoelectric focusing gel.

Preparation of Magnesium:RNA Outer Coordination (Ionic) Complex

The preparation for the ionic magnesium:RNA complex followed theprocedure for the covalent analog exactly except the stock magnesiumchloride solution was prepared in RNAse free water instead of anhydrousDMSO. The resultant colorless pellet was air-dried for several minutesbefore testing in the isoelectric focusing gel.

Preparation of RNA:Zinc Inner Coordination (Covalent) Complex

The preparation for the covalent zinc:RNA complex followed the procedurefor the magnesium analog exactly except a stock zinc chloride solutionwas prepared instead of a stock magnesium chloride solution. Theresultant colorless pellet was air-dried for several minutes beforetesting in the isoelectric focusing gel.

Preparation of Zinc:RNA Outer Coordination (Ionic) Complex

The preparation for the ionic zinc:RNA complex followed the procedurefor the covalent analog exactly except the stock zinc chloride solutionwas prepared in RNAse free water instead of anhydrous DMSO. Theresultant colorless pellet was air-dried for several minutes beforetesting in the isoelectric focusing gel.

Isoelectric Focusing Gel Experiment

This is a novel approach of using IEF gels to monitor for theanticipated modification of the iRNA target. FIG. 31 shows the resultsfrom the initial experiment.

Interpretation of IEF Experiments

The IEF experiment showed that magnesium RNA complexes prepared inanhydrous (A) conditions with three concentrations of magnesium chlorideproduced covalent complexes in approximately 50% yield. Magnesium RNAcomplexes prepared in aqueous (W) conditions with three concentrationsof magnesium chloride produced ionic complexes. The zinc RNA complexprepared in anhydrous (A) conditions with zinc chloride produced acovalent complex in approximately 50% yield. The zinc RNA complexprepared in aqueous (W) conditions with zinc chloride produced an ioniccomplex.

1-47. (canceled)
 48. A metal coordination complex of a biologicallyactive moiety and a metal, wherein the biologically active moiety isselected from the group consisting of: metformin, omeprazole,famotidine, rabeprazole, theophylline, hydrochlorothiazide, alendronate,mitoxantrone, adefovir, acyclovir, clonidine, dipyramidole, atovaquone,idarubicin, etidronate, valganciclovir, saquinavir, fosamprenavir,retinoic acid, minoxidil, cyclosporine, fosphenytoin, atrial natriureticpeptide, abarelix, and heparin and their derivatives.
 49. The complex ofclaim 48, wherein the metal is selected from the group consisting of:aluminum, bismuth, calcium, iron, strontium, magnesium, silicon, andzinc.
 50. A metal coordination complex of a biologically active moietyand a metal, wherein the biologically active moiety contains aβ-diketone, ketophenol or β-ketoalcohol, functional group.
 51. Thecomplex of claim 50, wherein the metal is selected from the groupconsisting of: aluminum, bismuth, calcium, iron, strontium, magnesium,silicon, and zinc.
 52. A metal coordination complex of a biologicallyactive moiety and a metal, wherein the biologically active moietycontains an azole functional group.
 53. The complex of claim 52, whereinthe metal is selected from the group consisting of: aluminum, bismuth,calcium, iron, strontium, magnesium, silicon, and zinc.
 54. The complexof claim 48, wherein the biologically active moiety is omeprazole. 55.The complex of claim 54, wherein the metal is selected from the groupconsisting of: bismuth, calcium, iron, strontium, magnesium, silicon,and zinc.
 56. The complex of claim 54, wherein the metal is selectedfrom calcium, strontium, and magnesium.
 57. The complex of claim 54,wherein the metal is calcium.
 58. The complex of claim 54, wherein themetal is magnesium.
 59. The complex of claim 54, wherein the metal isstrontium.
 60. A method of treating gastro esophageal reflux disease,comprising administering a complex of claim 54 to a patient in needthereof.